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Developmental pattern of alkaline phosphatase in soluble and particulate fractions of rat skull cap and femur
Abstract
Alkaline phosphatase is detected in both soluble and particulate fractions of rat bone. The concentration of alkaline phosphatase in the particulate fraction of rat skull cap bone and femur is high during the peak period of calcification of these bones suggesting the possibility of using it as a marker for the rate of bone calcification.
... The particular importance of restricting growth early in life may relate to the very high rate of growth that occurs during the ®rst few weeks of life (Marks, 1979), and the lower bone ash content early in life (Wise, 1970b). Murthy et al. (1986), working with rats, have suggested that feed restriction may work by reducing the growth rate of muscle tissues, thereby increasing the relative growth rate of the skeleton. This hypothesis is supported by the ®nding of Pinchasov et al. (1985), that feed restriction increased body ash content. ...
Leg disorders in broilers are a major economic and welfare problem. The aetiology of many disorders is complex but includes genetics, growth rate (due to feed restriction or lighting regime), feed conversion efficiency and body conformation, exercise, circadian rhythms, nutrition and stocking density. These categories are not mutually exclusive as one aetiology factor may affect another. Many studies of leg disorders fail to identify the specific pathological condition underlying the observed lameness. However, disorders may be classified according to underlying pathology as infectious, developmental and degenerative. This classification is difficult because these categories are also not mutually exclusive. Infectious conditions include bacterial chondronecrosis with osteomyelitis (BCO or femoral head necrosis, FHN), tenosynovitis and arthritis, infectious stunting syndrome (ISS) and viral induced neoplasia. Developmental conditions include varus valgus disease (VVD), rotated tibia, tibial dyschondroplasia (TD), rickets, chondrodystrophy and sondylolisthesis. Degenerative disorders include osteochondrosis (often TD), epiphyseolosis (often classified as FHN), degenerative joint disease (DJD), spontaneous rupture of the gastrocnemius tendon and contact dermatitis. BCO, TD, dermatitis and VVD are the most common disorders. Outbreaks of leg disorders are often site context specific. The welfare of broilers with leg disorders may be impaired due to pain from the condition, an inability to walk leading to frustration and associated problems of being unable to feed and drink due to immobility (which may result in starvation). In assessing welfare, the individual broiler must be considered irrespective of the frequency of occurrence of the disorder. Most studies of welfare in relation to leg disorders have used a subjective gait scoring method (0 is normal walking and 5 is unable to walk). Gait scoring is a practical method for assessing broiler lameness in the field. The method provides a useful tool to employ in the field without recourse to pathological investigation and, while the method conflates conformity with pathology, it is a helpful and constructive additional method to assist in welfare studies. For birds with scores greater than 3, lameness may be viewed as severe enough to potentially impair welfare. It is difficult to assess all disorders in relation to frequency of occurrence and their impact on welfare due lack of evidence. BCO (or FHN and BCN) is the most common disorder and is often severe in form. TD (incl. epiphyseolysis) and rickets are common, often sub-clinical but when severe there is a considerable impact on welfare. Contact dermatitis may be common under certain conditions and causes poor welfare when severe. Gastrocnemius tendon slippage, tenosynovitis, DJD and spondylolisthesis are not so common but are likely to cause poor welfare when they occur (i.e. pain and prevention of certain behaviours). VVD and rotated tibia can be common but tend not to be directly painful unless another condition is present. However, they can cause poor welfare if the bird is not culled and lead to an inability to walk, feed, drink and perform other behaviours. ISS, viral induced neoplasia and chondrodystrophy have only a small impact on welfare in the UK flock, the last because it is no longer encountered. To decrease the prevalence of leg disorders, growth rate needs to be decelerated: meal feeding, feed restriction during the early period of rearing, lower stocking densities and increased activity can result in a considerable reduction in leg problems. Continuous lighting should be avoided; overall the light period should be reduced. There also needs to be careful management of litter to prevent dermatitis. Factors in the diet are also important, particularly Ca, P and D3 which can prevent certain disorders (e.g. TD). Further research is needed to establish the prevalence of disorders, underlying pathology in relation to subjective gait scores and analgesics in relation to pain mechanisms. A forum for discussion of these issues should be established and further control-trials conducted to investigate these factors.
... Although significant quantities of APs are present in different tissues, their physiological roles are still unclear, except that of bone origin. It is known, that enzyme level reflects the activity of osteoblasts and that it is involved in the formation and mineralization of the bone matrix (Rodan & Rodan, 1984;Wuthier & Register, 1985;Ali, 1986;Murthy et al, 1986). Hence, increased enzyme activities are observed during normal growth of bone as well as in development of neoplastic and allied osteoproliferative disorders (Jaffree & Price, 1965;Ingleton et al, 1979;Ghanta et al, 1980). ...
Alkaline phosphatase (AP) activities of sera from guinea fowl infected with osteopetrosis virus strain PTS-56 were investigated. Enzyme activities in birds of infected and control groups varied. AP activities in control guinea fowls, 10 to 15 weeks of age, were twice as high as those of 1-year-old birds whose growth was completed. A positive correlation was found between the intensity of virus-induced excessive bone growth and serum AP activity, which was 628 +/- 68.9 microM by the 17th week and reached 1283 + 76.8 microM by the 30th week post-infection (p.i.). Later AP activity decreased, but even at 52 weeks p.i. it was higher than that of untreated guinea fowl. The presumed high proportion of AP of bone origin of the total serum enzyme activities was supported by the low values of residual AP activity after heat inactivation at 58 degrees C (14.7 +/-3.7%) and after precipitation with wheat germ lectin (13 +/-1.2%) during the period of active bone tumour formation.
Seven hundred and twenty one-day-old AA broiler chickens were randomly allocated into two groups (male and female for half), and put into two identical closed houses with different lighting programs. The first house was illuminated by using common incandescence light, and the second one was added with ultraviolet radiation light from the second week onwards. The birds lived in a floor with litters and free access to feed and water. Temperature, humidity and immune programs in the two houses were similar. The results showed that under ultraviolet radiation, the growth speed of skeleton increased (the shank length was significantly increased in the third week, P
An 8-h photoperiod, compared with 12-, 16- or 23-h photoperiods, has been shown to reduce the incidence of injurious pecking and leg problems in intact male turkeys but to result in lower body weights, slower sexual development and less efficient food conversion. The effect of 8 h of illumination given as a solid photoperiod, as eight 1-h periods equally spread through the 24 h or concentrated into a 12-h part of the day on the performance of BUT 8 intact male turkeys was investigated.
Fragmentation of the daily illumination increased body weight, improved food conversion efficiency but did not significantly affect food intake. Increased sexual displaying and significantly better food conversion between 16 and 20 weeks by the two intermittently illuminated groups strongly suggested that both regimens were interpreted as stimulatory daylengths. Generally, losses due to mortality and culling were higher in both fragmented groups, while injurious pecking was higher in 8(1L: ID) birds before 5 weeks and in birds given a 12-h ‘subjective day’ after 5 weeks than in 8-h controls. Culling due to leg problems was significantly higher in birds on the 8(1L:2D) regimen but the incidence of leg imperfections was minimal (< 0·037) in all groups. Both intermittent groups but the 8(1L: 2D) in particular, showed a reduced response to hand movement across the eyes compared with 8-h controls. The controls ate proportionately more food during their 16-h dark period than did the intermittent group during their 12-h ‘subjective night’, though both groups increased their ‘nocturnal’ feeding with age.
Objective: An in vitro assessment of MG-63 human osteosarcoma cells' alkaline phosphatase (ALP) activity when in contact with calcium hydroxide powder (CH), paste (CHP) and grey mineral trioxide aggregate (MTA). Methods: MG-63 cells were seeded to the three selected materials for durations of 0.25, 0.5, 1, 24, 48 and 72 hours. BCIP-NBT assay was used and ALP activity quantified using ELISA reader at 410 nm. Results: The overall analysis for ALP activity indicated significant interaction between test materials and control (maintenance medium). Subsequently, the test materials were paired and analysed for initial (0.25, 0.5, 1 hour) and delayed response (24, 48 and 72 hours). During the initial response, CH exhibited an increased ALP activity compared to MTA. This interaction was not dependant on duration. The delayed response exhibited elevated ALP activity with CHP when compared to MTA and CH. The interaction of CHP was dependant on duration. Conclusion: All three materials exhibited increased ALP activity.
In the femoral extremities of the adult rat containing the metaphysis, the epiphyseal cartilage, and the epiphysis, four alkaline phosphatase (AP) forms were distinguished on polyacrylamide gel electrophoresis. Two soluble forms were present in the 160,000 g supernatant: one of Mr 165 kDa and another of Mr 110–115 kDa, which exhibited a strong catalytical activity. Moreover, from the pellet, three membrane-bound forms of Mr 130, 110–115, and 100 kDa could be extacted with sodium deoxycholate. When denaturated AP was visualized by postelectrophoretic autoradiography of the phosphorylated intermediates, subunits always appeared as three monomers of Mr 75–80, 60–70, and 50–60 kDa. As four native forms but only three types of subunits were found to be present in the femur, it seems that, apart from homodimers, some heterodimers could also occur. Three types of diets were administered to three groups of rats for 5 weeks. Two are known to disturb bone mineralization: (1) a vitamin D3-deficient diet, and (2) the same as (1) but enriched with 12% sorbitol. The third was a normal diet containing vitamin D3. Concerning the effects on AP of dietary sorbitol and the vitamin D3-deficient diet, it was found that rats receiving the diet supplemented with sorbitol showed a substantial rise in the activity of the Mr 165 kDa form with the concomitant appearance of a new monomer of Mr 100 kDa. In contrast, rats fed the vitamin D3-deficient diet always displayed an increase in enzyme activity, principally of the Mr 100 and 110 kDa forms. In conclusion, the femur extracts of normal rats contained different forms of AP: either soluble 110–115 and 165 kDa forms or membrane-bound 130, 110–115, and 100 kDa forms. The administration of sorbitol-enriched diet induced a marked increase of the 165 kDa form whereas the administration of vitamin D3-deficient diet increased the 100 and 110 kDa forms.
The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bone (Silbermann, M., D. Lewinson, H. Gonen, M. A. Lizarbe, and K. von der Mark. 1983. Anat. Rec. 206:373-383). We have studied the expression of genes that typify osteogenic differentiation in mandibular condyles during in vitro cultivation. RNAs of the genes for collagen type I, osteonectin, alkaline phosphatase, and bone gla protein were sequentially expressed in progenitor cells and hypertrophic chondrocytes during culture. Osteopontin expression peaked in both the early and the late phase of the differentiation process. The data indicate a distinct sequence of expression of osteoblast-specific genes during osteogenic differentiation and new bone formation in mandibular condyles.
Tenascin is a glycoprotein of the extracellular matrix, which has been associated with differentiation of hard tissue forming cells. Alkaline phosphatase (AP) is involved in calcification, and it has also been suggested to function in cell differentiation. We have compared the distributions of tenascin and AP in the developing skull and teeth of embryonic and growing rats and mice. Tenascin was localized by immuno‐Peroxidase and AP by enzyme histochemical staining of tissue sections. Both tenascin and AP were largely restricted to bone, cartilage, and teeth. In cartilage, tenascin was expressed in the perichondrium, whereas AP activity was detected only in the hypertrophic cartilage. In growing intramembranous bone, tenascin and AP were expressed in the periosteum and endosteum. AP activity was restricted to the inner layer of the periosteum, whereas tenascin expression extended to the more superficial layers. In bud‐staged teeth tenascin but no AP activity was localized in the condensing mesenchymal cells around the epithelial bud. At the bell stage both tenascin and AP activity were localized in the cuspal mesenchyme, and the intensity of staining decreased towards the cervical region.
In summary, tenascin was present at all sites of AP activity except in the epithelial cells of the enamel organ and the hypertrophic cartilage of the mandibular condyle. In mesenchymal tissues tenascin was more widely distributed than AP. It can be suggested that tenascin has functions at earlier stages of hard tissue formation than AP.
The rat bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) gene is expressed at high level in these particular tissues and at low levels in many other tissues. To study the mechanisms underlying the complex regulation of the rat BLKP ALP expression, we isolated a genomic clone, containing a 10.5-kb insert, which includes the promoter of the BLKP ALP gene with 2kb of 5' flanking region, its first exon (84bp), and over 7kb of the first intron. The promoter of the rat BLKP ALP displays features of a “housekeeping” gene promoter: an atypical TATA-box (TTCATAA); 3 potential Spl binding sites; high GC content (82% in positions -134 to -14); and a high CpG to GpC ratio (60:89 in the 0.85-kb promotor region), indicating an abundance of potential methylation sites. Likewise, transient transfection of CAT fusion genes into ROS 17/2.8 osteoblast-like cells reveals weak expression from the promoter and proximal 5' flanking sequences, which can be elevated by an SV40E enhancer.
We studied the effects of undernutrition during the suckling period on the development of the calvarium and the femur of rat pups suckling dams fed a 20% or 5% protein diet. The results show that the maturation of bone is delayed by undernutrition. In both the calvarium and the femur, the particulate fraction alkaline phosphatase reflects the rate increments in calcium and phosphorus uptake in control animals. This association is affected in the undernourished pups, as both the alkaline phosphatase activity in particulate fraction and the calcium and the phosphorus uptake decreased. However, the extent of disturbance was more prominent in the femur than in the calvarium, suggesting that during postnatal undernutrition the calvarium is less affected when compared to prenatal undernutrition, where the femur is less affected. The results from these studies suggest that proper nutrition is necessary for bone development during both the prenatal and postnatal period.
We studied the effect of maternal protein deficiency on the development of the rat calvarium and femur. The results show that maternal protein deficiency during gestation leads to fetal growth retardation, and that the calvarium is more affected than the femur. A significant decrease in the activity of alkaline phosphatase was found in the soluble fraction in both the calvarium and the femur. However, this was not true in the case of the particulate fraction in which a significantly increased activity was found. This increase was not associated, however, with a concomitant increase in calcium and phosphorus (per 100 g fresh bone).
Lumbar vertebrae (L4) from CW-1 female mice were examined for age-related changes in alkaline and acid phosphatase activities from young to old age. Histochemically, both enzymes were encountered along the bony surfaces of both trabecular and cortical bones with no significant age-related changes in their distribution. Biochemical determinations of bone alkaline phosphatase (Alk'ase) activity revealed that for a given unit level of bone or the bone as a whole no significant changes took place, whereas acid phosphatase (Acid'ase) activity was found to have increased significantly with age. A high positive correlative relationship was noted between the calcium content and the trabecular bone volume of the same vertebrae. It may, therefore, be proposed that age-related bone loss in mice could be attributed to an enhanced resorption rather than to a substantial reduction in the formative potential of bone cells.
1. Activities of alkaline phosphatase, liver-membranous, liver-soluble and serum-soluble, were dramatically induced in dogs by treatment with both phenobarbital and brovanexine. The treatment induced a 17-fold increase in membranous, a 155-fold increase in soluble, and a 105-fold increase in serum alkaline phosphatases. 2. There was no difference in the enzymatic behavior of the three forms of alkaline phosphatase, on heat stability, amino acid inhibition and optimum pH. 3. When the three alkaline phosphatases were treated initially with n-butanol, their apparent molecular size was identical. After treatment with phosphatidylinositol-specific phospholipase C, the liver-soluble and serum-soluble alkaline phosphatase were of the same molecular size. Liver-membranous alkaline phosphatase, however, was larger in molecular size than the other two forms, suggesting a difference between soluble and membranous alkaline phosphatase forms. 4. In terms of the sugar moiety of the three alkaline phosphatase forms, the membranous enzyme showed more of the higher affinity fraction and less of the lower affinity fraction of concanavalin A, compared with the soluble enzymes. 5. Consequently, it is possible that the membranous enzyme may be solubilized by an enzyme such as phosphatidylinositol-specific phospholipase C and modify further the sugar moiety of alkaline phosphatase molecules, resulting in serum alkaline phosphatase transfer from the soluble enzyme in liver.
Characteristics of human renal cortex and medulla alkaline phosphatase (ALP) were compared. Enzymatic and hydrophobic properties of both ALPs were almost similar. However, the results of concanavalin A affinity chromatography and wheat germ agglutinin affinity electrophoresis, exhibited that sugar chain structure(s) might be different between the cortex and medulla ALPs. In addition, the molecular mass and substrate specificity differed from each other, and these results of cortex and medulla ALPs were well accordant with those of human duodenal and ileal ALPs, respectively, as described previously (Clin Chim Acta 1987;163:279-287).
Females are often supposed to have a lighter skeleton than males, even in avian domestic species. However, in broiler chickens, females are less susceptible to bone deformities than males. In order to better understand these conflicting facts, male and female broilers were compared for the growth of cortical bone. Morphology, histomorphometry, composition and biomechanical properties of the tibiotarsi were analysed in both sexes at 1, 12, 26 and 42 days of age. The quantity of bone tissue of the tibiotarsus (weight, volume, diameter of the diaphysis, area of the cortex) was smaller in females, although the occurrence of varus-valgus deformations of the intertarsal joint was largely reduced in female chickens (8.8% versus 19.9% in males at 42 days of age). The tibia became significantly lighter in females from 26 d of age. Differences in tibia length and volume became significant at 42 d of age only, while cross-sections of the diaphysis were smaller in females from the hatching, leading to thinner bones in females. The percentage of dry matter of tibiae was higher in females from hatching. From 12 days old, tibiotarsi of females tended to be less porous and were more mineralized (higher ash/dry matter ratio). In females, mineralization proceeded at a higher rate (MAR) until 26 d of age and MAR became higher in males afterwards. The stiffness of the tibia diaphysis was similar in males and females all along the growth. In conclusion, the growth of cortical bone is very different in male and female broilers. In female broilers, the thinness of bone diaphysis is counter-balanced by modifications in the composition of the matrix and in the porosity of the cortex, leading to equal biomechanical characteristics of tibiotarsi in both sexes.
The activities of alkaline and acid phosphatases, the linear dimensions and volumes of mandibles and long bones, and the level of calcitonin in serum of rat pups suckled on dams fed 25 or 6% protein diets were determined. In both mandibles and long bones, alkaline phosphatase activity corresponded to the calcification pattern, and acid phosphatase activity paralleled organic matrix formation. The calcitonin level in blood serum of the malnourished pups differed from that of controls only on day 5. The linear dimensions and volume were consistently more affected in long bones than in mandibles. These results may be explained in terms of the critical growth periods of these bones. In the mandible, the critical growth period appears to occur prenatally, whereas in the long bone it occurs postnatally, when the nutritional stress of this experiment was applied. Long bones were thus generally more affected than mandibles.
Collagen synthesis, 45Ca uptake, and the half-life of 45Ca in mandibles and long bones of rat pups suckled on dams fed 25 or 6% protein diets were determined. Collagen synthesis in both mandibles and long bones was impaired in the malnourished group at an early age. Protein-energy malnutrition appears to affect different developmental processes in these two bones. The principal interference with collagen synthesis in the mandible occurs when bone proline is converted into hydroxyproline. In contrast, proline uptake from the blood for deposit in bone matrix is the step of collagen synthesis mainly affected in long bone. In addition, the critical growth period of mandible was different from that of long bone. The calcium complex fraction (calcium bound to protein plus that in inorganic salts) was the main compartment affected in the malnourished group. However, the efficiency of calcification per milligram of matrix in the calcium complex fraction of the mandible and long bone was approximately the same in the control and malnourished groups, thus suggesting that accumulation of calcium occurs in parallel with the formation of bone matrix regardless of the nutritional conditions. The half-life of 45Ca in the various calcium fractions of both types of bone was 72 hours in both the control and malnourished groups except the calcium complex portion of the long bone of the control group, which was about 100 hours.
In a previous communication (Li, H.-C., Hsiao, K.-J., and Chan, W.W.S. (1978) Eur. J. Biochem. 84, 215-225) we reported the purification of a divalent cation-independent, nonspecific phosphoprotein phosphatase (phosphatase S, M(r) = 35,000) from canine cardiac muscle to apparent homogeneity. It was found that the homogeneous enzyme preparation exhibited significant activity toward p-nitrophenyl phosphate. since the enzymic activity had an optimum pH around 8.5, it was termed alkaline phosphatase S. Further studies demonstrated that the major alkaline phosphatase activity in the soluble fraction of the canine heart homogenate was co-purified with the phosphoprotein phosphatase S throughout the purification procedure. The alkaline and the phosphoprotein phosphatase activities were found to co-migrate on polyacrylamide gel electrophoresis, ion exchange, and gel filtrtion chromatographies and sucrose density gradient ultracentrifugation. These two activities, however, exhibited distinct thermostability, pH activity profile, and metal ion specificity, and could be partially separated by hydrophobic interaction chromatography. Discussions concerning whether these two activities reside in the same enzyme protein or in two different yet very similar polypeptide chains have been presented. The properties of alkaline phosphatase S appear to be different from other known mammalian alkaline phosphatases, and it may represent a new isozyme species of the hydrolytic enzyme. In contrast to other well studied alkaline phosphatases of membrane origin purified from various tissues, the cardiac muscle alkaline phosphatase S was of cytosolic origin and required the simultaneous presence of Mg2+ and a sulfhydryl compound for activity. The close association of alkaline phosphatase S activity with phosphoprotein phosphatase S suggests that it may play a role in the regulation of protein phosphorylation-dephosphorylation reaction.
Several parameters were measured in the growth centers of mandible and long bone of rat pups suckled on dams fed 25% or 6% protein diets. No sex-related differences were found in any parameter either during normal growth or in response to malnutrition. While the total contents of protein, DNA, RNA and calcium were reduced in both tissues, mandible was less affected than long bone, although it appeared to be affected earlier. Moreover, malnutrition reduced the number of cells in the mandible without changing cell size or calcium content. In contrast, long bone appeared to be affected later than mandible, and to be modified differently. In this case, not only cell number, but also cell size and cellular content of calcium were reduced. The results can be interpreted to mean that the critical periods of growth for mandible and long bone are different. Moreover, some suggestion of differences in relative sensitivity to malnutrition of these two hard tissues was also observed.
Transplantation of collagenous matrix from the rat diaphyseal bone to subcutaneous sites resulted in new bone formation by an endochondral sequence. Functional bone marrow develops within the newly formed ossicle. On day 1, the implanted matrix was a discrete conglomerate with fibrin clot and polymorphonuclear leukocytes. By day 3, the leukocytes disappeared, and this event was followed by migration and close apposition of fibroblast cell surface to the collagenous matrix. This initial matrix-membrane interaction culminated in differentiation of fibroblasts to chondroblasts and osteoblasts. The calcification of the hypertrophied chondrocytes and new bone formation were correlated with increased alkaline phosphatase activity and 45Ca incorporation. The ingrowth of capillaries on day 9 resulted in chondrolysis and osteogenesis. Further remodelling of bony trabeculae by osteoclasts resulted in an ossicle of cancellous bone. This was followed by emergence of extravascular islands of hemocytoblasts and their differentiation into functional bone marrow with erythropoietic and granulopoietic elements and megakaryocytes in the ossicle. The onset and maintenance of erythropoiesis in the induced bone marrow were monitored by 59Fe incorporation into protein-bound heme. These findings imply a role for extracellular collagenous matrix in cell differentiation.
Alkaline phosphatase has been purified from microsomes of chicken epiphyseal cartilage by first selectively extracting certain adventitious proteins with 0.25 M trichloroacetate. The membrane-bound enzyme was then solubilized by 1% cholate in buffered 33% saturated ammonium sulfate and purified by column chromatography on Bio-Gel A-5m, extraction with 1-butanol, and ion exchange chromatography on DEAE-Bio-Gel A. The purified alkaline phosphatase from the cartilage membrane had a subunit molecular weight of 53,000 and a holoenzyme weight of 207,000-220,000, indicating a tetramer. The pH optima for p-nitrophenylphosphate, ATP, and pyrophosphate hydrolysis were 10.3, 9.0, and 8.5, respectively. Values of Vmax (in micromoles/min/mg) were 220, 3.1, and 0.8, respectively. Substrate inhibition was pronounced at values of pH below 8.5. Inhibition of p-nitrophenylphosphate hydrolysis at pH 10.3 showed that phosphate and arsenate were competitive inhibitors (KI = 1.88 and 0.15 mM, respectively) and levamisole was an uncompetitive inhibitor (KI = 0.32 mM), while L-phenylalanine and ZnCl2 were mixed inhibitors (KI = 15.8 and 0.02 mM, respectively). Inhibition by preincubation in 1 mM EDTA was reversible by readdition of 0.25 mM MgCl2 nd 20 microM ZnCl2. The data indicate that this membrane-bound alkaline phosphatase from chicken epiphyseal cartilage is a Zn2+ and possibly Mg2+-containing enzyme. While the subunit molecular weight and kinetic properties of the enzyme are quite typical of vertebrate alkaline phosphatases, the tightness of binding to the membrane lipids, the extreme sensitivity to substrate inhibition, and the tetrameric conformation of the holoenzyme are unusual.
Enzymically active intestinal alkaline phosphatase exists in both soluble and membrane-bound forms in the suckling rat. Antiserum prepared against purified soluble alkaline phosphatase (anti-AlP) was shown to be monospecific when assessed by Ouchterlony double-diffusion analysis and immunoelectrophoresis. The two forms of alkaline phosphatase were antigenically identical and possessed similar affinities for anti-AlP. To study the biosynthesis of the two forms, 14-day-old rats were injected intraperitoneally with [(3)H]leucine. The labelling kinetics of alkaline phosphatase, extracted from supernatant and brush-border membrane fractions with anti-AlP, was followed over 20h. Incorporation of [(3)H]leucine into membrane-bound alkaline phosphatase was rapid, reaching a plateau at 6h. The soluble enzyme showed slower incorporation of label and maximal radioactivity was not reached until 12h after labelling, a lag of 6h behind the membrane-bound enzyme. Soluble alkaline phosphatase could not have been a precursor of the membrane form, as there was no early peak of radioactivity in the soluble form. To determine if the soluble enzyme was irreversibly derived from the membrane enzyme, a newly developed technique of labelling brush-border membrane proteins in vivo by intraluminal injection of diazotized [(125)I]iodosulphanilic acid was used. The appearance of (125)I in soluble and membrane alkaline phosphatase was then monitored over a 7h period, encompassing the lag between maximal leucine labelling of the two forms. The results failed to show either a proportional transfer of radioactivity from membrane to soluble alkaline phosphatase or an absolute increase in radioactivity of the soluble form during degradation of brush-border alkaline phosphatase. Therefore there does not appear to be a serial precursor/product relationship between the soluble and membrane-bound forms of suckling-rat intestinal alkaline phosphatase.
Many of the properties unique to the calcified tissues of vertebrates can be ascribed to the mineral salts investing the interstitial spaces of these structures. By far the most distinctive of these properties, hardness, can be attributed directly to the ultra-structural interrelations between the mineral and the organic, fibrous extracellular matrix. To many, however, this hardness implies inertness and passivity, especially of the mineral component itself. But, the mineral salts of bone are far from chemically inactive, for in addition to their mechanical function, these salts play an important role in the dynamic physiological chemistry of living hard tissue. It is this dual mechanical and physiological character that makes the study of the mineral component of bone a fascinating subject; and it is challenging to try to understand how the mineral can fulfill its two disparate, yet complementary roles. This chapter will attempt to summarize the most recent progress in the description of this component and to review the current concepts concerning its origin and its physiological-structural functions. Emphasis will be placed on the mineral itself, alluding to the organic and cellular components only in reference to their involvement in the formation, maintenance, and function of the mineral component.
Cell differentiation is one of the central problems of contemporary biology and medicine. How does the fertilized egg develop into an adult organism with a myriad of cell types? It is common knowledge that all cells in a multicellular organism possess the same genetic information, hence an identical genotype. What is the molecular basis of the progressive appearance of diverse phenotypes from cells with the same genotype? The alteration of gene expression during development is of cardinal importance in cell differentiation.
The specific activity, tissue specificity, and subcellular distribution of alkaline phosphatase were studied in the fetal
rat limb during initial cartilage calcification and bone formation. The pH optimum, Km, activation, and inhibition characteristics
of the enzyme assayed for in 900 ×g supernates of whole limb homogenates indicated that the activity represented a fetal bone alkaline phosphatase. Studies examining
temporal changes of the enzyme in these preparations demonstrated a substantial increase in activity over each of the days
during which they were studied (days 15–18). Fractions derived from the discontinous density gradient centrifugation of the
limb preparations were used to study the chronological subcellular distribution of the enzyme. Enzyme activity was found in
all of the fractions with the greatest activity occurring in fractions consisting of ribosomes and small vesicles. The vesicular
component was similar to the matrix vesicles described by others in calcifying tissues. The daily increase in activity measured
in the crude supernate was further reflected in the distribution studies. The association of alkaline phosphatase with the
vesicular structure is compatible with the theorized functions of matrix vesicles, and the substantial increase in activity
between days 15 and 18 further demonstrates an intimate association of alkaline phosphatase with skeletal development.
Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg2+, Ca2+, and PPi. Further, the ATPase activity was not activated by Ca2+ in the presence of an optimal Mg2+ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PPi, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.
Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix-vesicle fraction were studied in detail.Mg2+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent.Ca2+ stimulated all activities weakly in the absence of Mg2+. However, when Mg2+ was present, Ca2+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca2+, and hence would not seem to be involved with active Ca2+ transport in the typical manner.The distribution of alkaline phosphatase, pyrophosphatase, and Mg2+-ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na2+ + K+)-ATPase activity was not found in the cell fraction. All activities showed nearly identical sensitivities to levamisole (4 · 10−3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10—15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg2+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.
1.1. The proteins of the intestinal microvillus membrane have been studied during post-natal development in the rat (days 12–37).2.2. In suckling animals (up to age 20 days), the majority of alkaline phosphatase, glucoamylase and lactase activities in the distal half of the intestine were located in the supernatant fraction (). These enzymes were attached to the membrane from the proximal intestine at all ages.3.3. Alkaline phosphatase, maltase and lactase activities in the supernatant fractions chromatographed in Sephadex G-200 in positions similar to the corresponding membrane enzyme. Corresponding activities for lysosomal counterparts of maltase and lactase present in the supernatant fraction chromatographed differently. Moreover, pH optimum of the soluble enzymes was 9.2 for phosphatase and 5.5–6.0 for glycoamylase and lactase. The soluble lactase and alkaline phosphatase were inhibited minimally by , and sodium fluoride respectively. L-Phenylalanine (20 mM) did inhibit the soluble phosphatase by 90%. Thus, the soluble enzymes are not mainly of lysosomal origin, but have characteristics of membrane-bound enzymes.4.4. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed 18 protein bands which were present in adult membranes. Two other proteins were unique for membranes of distal intestine in suckling rats. The proteins corresponding to known enzyme activity changed as expected with age (e.g. sucrase, maltase increased, lactase decreased). Most of the other proteins were also altered in amount during development. Thus, the changes in the microvillus membrane during development in the rat are not limited to specific enzymes.
Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg2+, Ca2+, and PPi. Further, the ATPase activity was not activated by Ca2+ in the presence of an optimal Mg2+ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PPi, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.
The specific activity, tissue specificity, and subcellular distribution of alkaline phosphatase were studied in the fetal rat limb during initial cartilage calcification and bone formation. The pH optimum, Km, activation, and inhibition characteristics of the enzyme assayed for in 900 X g supernates of whole limb homogenates indicated that the activity represented a fetal bone alkaline phosphatase. Studies examining temporal changes of the enzyme in these preparations demonstrated a substantial increase in activity over each of the days during which they were studied (days 15-18). Fractions derived from the discontinuous density gradient centrifugation of the limb preparations were used to study the chronological subcellular distribution of the enzyme. Enzyme activity was found in all of the fractions with the greatest activity occurring in fractions consisting of ribosomes and small vesicles. The vesicular component was similar to the matrix vesicles dexcribed by others in calcifying tissues. The daily increase in activity measured in the curde supernate was further reflected in the distribution studies. The association of alkaline phosphatase with the vesicular structure is compatible with the theorized functions of matrix vesicles, and the substantial increase in activity between days 15 and 18 further demonstrates an intimate association of alkaline phosphatase with skeletal development.
Epiphyseal cartilage from rat tibia was treated by the ruthenium red method and lead citrate method to demonstrate the presence and distribution of alkaline phosphatase, examined by electron microscope. Enzyme activity first appeared as electron dense precipitates at the plasma membrane of chondrocytes in the proliferative zone. No precipitates were found in the intercellular matrix. In the upper part of the hypertrophic zone, electron dense precipitates were seen at the plasma membrane of chondrocytes and in association with small spherical or ovoid structures with a diameter of 800–1,200 Å which were present around chondrocytes and in the intercellular matrix. These structures were not enveloped by unit membranes. Moreover, reaction products were adherent to the outersurfaces of the investing membrane of matrix vesicles within the longitudinal septum. In the lower hypertrophic zone, reaction products were found at the plasma membrane of chondrocytes and in association with spherical or ovoid structures with a diameter of 1,000–2,000 Å which were observed around chondrocytes. In areas of advanced calcification, the reaction products around chondrocytes were in smaller quantities, but they were seen in association with variously shaped bodies with a diameter of 2,000–6,000 Å which were found within the longitudinal septum. Some of these bodies contained needle-like crystals. The possible role of the intercellular activity of alkaline phosphatase was discussed.
Summary Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm3 band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm3 band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm3 band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm3 band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm3 fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation withβ-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.
Coarse powders of acid-insoluble matrix of diaphysis and calvarial parietal bone rapidly and consistently transformed fibroblasts into masses of cartilage and bone containing hemopoietic marrow. The transformant was encapsulated by fibroblasts within 24 hr to form a plaque. Transformation was restricted to the central thicknesses of the plaque. Under the stated conditions the alteration of the phenotype, fibroblast to chondroblast, was an unstable transformation, whereas the phenotype change, fibroblast to osteoblast, was stable. The transformation occurred on a rigid timetable of sequences. Measurements of alkaline phosphatase activity and incorporation of radioactive sulfate, phosphate, and calcium were sensitive and quantitative assays for the appearance of the transformed products, cartilage and bone.
The pyro- and alkaline phosphatase activities of quail bone appear to be catalyzed by a single enzyme. The activities could not be separated after ion-exchange chromatography or gel filtration. The pH optima for pyro- and alkaline phosphatase were 8.0 and 10.7, respectively. Alkaline phosphatase activity was stimulated by magnesium. Optimal stimulation of pyrophosphatase activity occurred at a pyrophosphate to magnesium ratio of 2.5. Both enzymatic activities were not markedly affected by high concentrations of fluoride.
A serial longitudinal study was undertaken to determine the activities of the alkaline and acid phosphatases in the long bones and mandibles. The optimum pH of the two enzymes was recorded at 10.2 and 5.4 for alkaline and acid phosphatase, respectively. Synchronized and randomized litters of rats were killed, 1 litter daily, starting at day 1 to day 25 post partum. Samples were analyzed for protein concentration and activity of the phosphatases. A pattern of low and high activity was observed in both bony tissues, as well as a pattern of low alkaline phosphatase activity during acid phosphatase activity peaks, and vice versa.
The observed peaks suggest a correspondence between phosphatase activity and the other biochemical changes occurring in the growing bone, i.e., organic matrix and mineral formation. A separate study, considering the possibility of rhythmic features of the enzyme activity suggests that there may be a small diurnal rhythm at an early age.
Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5′-AMPase (EC 3.1.3.1., 3.6.1.1, 3.6.1.3, and 3.1.3.5, respectively). Most of the acid phosphatase (EC 3.1.3.2) activity, on the other hand, was found in the cellular fractions, indicating that matrix vesicles are quite distinct from lysosomes. This appears to be the first instance of isolation of membrane-bounded extracellular particles from any normal tissue.
The matrix vesicles possess enzymes that can increase the local concentration of orthophosphate and thus could lead to the formation of hydroxyapatite. The membrane-bounded matrix vesicles may also provide a mechanism for ATP-dependent transport of calcium or phosphate into the lumen of the vesicles with resultant mineralization.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
An enzyme is present in the ossifying cartilage of young rats and rabbits, which rapidly hydrolyses hexosemonophosphoric acid, yielding free phosphoric acid. In respect of this enzyme the kidney is considerably less active (about 50%) than an equal weight of epiphyseal cartilage. Other tissues contain the enzyme in a very much lower degree, muscle and blood being almost inactive. Non-ossifying cartilage shows less than one-tenth of the hydrolytic power of ossifying cartilage. The same tissues in approximately the same order also hydrolyse glycerophosphoric acid. One of the two phosphoric acid groups of hexosediphosphoric acid is very readily hydrolysed by almost all tissues (including muscle and non-ossifying cartilage) except blood. The possible significance of this enzyme in the process of ossification in the animal body is discussed and certain preliminary experiments in various directions are briefly described, by the further prosecution of which it is hoped to obtain more information on this subject.
Studies on chemical composition of bones in the rat in relation to nutritional factors A study of some aspects of reproduction by means of chemical analysis
- Gomez
- Mp
Gomez MP (1983) Studies on chemical composition of bones in the rat in relation to nutritional factors. Ph.D. thesis, M.S. University, Baroda 26. Spray CM (1950) A study of some aspects of reproduction by means of chemical analysis. Br J Nitr 4:354-360
The growth and development of cartilage and bone Biochemical development of the fetus and neonate
- Ah Reddi
Enzymes of cells in bone
- G M Jeffree
- GM Jeffree
Matrix vesicles of cartilage and bone The biochemistry and physiology of bone
- Hc Andreson
Studies on the composition of the bone in relation to age and nutritional status
- J R Dave
- JR Dave
Effects of deficiencies of food energy, protein and vitamin A on the composition of the femur in rats
- R Rajalakshmi
- Jr Dave
Studies on chemical composition of bones in the rat in relation to nutritional factors
- M P Gomez
- MP Gomez
Structure and chemistry of bone mineral. Cellular and molecular aspect Biological calcification
- Ed Eanes
- As Posner
The growth and development of cartilage and bone
- A H Reddi
- AH Reddi
Matrix vesicles of cartilage and bone
- H C Andreson
- HC Andreson
Colorimetric determination of phosphorus
- C H Fiske
- Subba Row
- CH Fiske