Biomedica biochimica acta (Biomed Biochim Acta )

Publisher: Akademie der Wissenschaften der DDR. Presidium; Deutsche Gesellschaft für Experimentelle Medizin


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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Hepsin is a membrane bound protease of 51 kDa present in mammalian cells. It contains a stretch of hydrophobic sequence of 27 amino acid residues in its N-terminal region. By employing fluorescent immunostaining of cells and western blot analysis of the various cell subfractions, the catalytic subunit (carboxyl terminal half) of hepsin is found at the cell surface. The hepsin gene is expressed in most tissues of a young adult baboon, particularly in the liver at high levels.
    Biomedica biochimica acta 02/1991; 50(4-6):791-3.
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    ABSTRACT: Two cystatins occur in mature seeds of the rice, Oryza sativa L. japonica, which are named oryzacystatin I (OC-I) and oryzacystatin II (OC-II). These are highly homologous to each other and are significantly homologous to cystatin superfamily members of animal origin, especially to family-2 cystatins. However, both lack disulfide bonds as in the case of family-1 cystatins (stefins). Each of OC-I and OC-II thus seems to be chimerical of family-1 and family-2 cystatins, and we propose that a new category such as "phytocystatin" be opened for these cystatins of plant origin. For specificity it was observed that OC-I inhibits papain 100 times more efficiently than cathepsin H, whereas, OC-II inhibits cathepsin H 100 times more efficiently than papain. A cysteine proteinase, named oryzain alpha, exists in germinating rice seeds. cDNA cloning studies have disclosed that two other related species, named oryzain beta and gamma, are also present. In respect to the amino acid sequence, oryzain alpha and beta are homologous to papain and oryzain gamma is homologous to cathepsin H. These observations suggest the possibility that either or both of oryzain alpha and beta are target enzymes of OC-I and oryzain gamma is a target enzyme of OC-II.
    Biomedica biochimica acta 02/1991; 50(4-6):637-41.
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    ABSTRACT: The increasing knowledge about specific human genes and their alterations causing genetic diseases, requires new strategies and methods for mutation-detection. However, the large molecular heterogeneity of such mutations in each gene sets serious limitations to this approach. The polymerase chain reaction has become the basis of several new developments aiming to overcome this problem. Recently developed methods to search for unknown mutations are the denaturing gradient electrophoresis, chemical mismatch cleavage, single stranded conformation polymorphisms and direct genomic sequencing. Known mutations in a particular gene can be detected by the allele specific hybridization, the amplification refractory mutation system, the competitive oligonucleotide priming reaction or the oligonucleotide ligation assay. As spin-off of the human genome sequencing projects, automated DNA-sequencing may in the future become the easiest, fastest, most reliable and most informative technique and a powerful day-to-day method in every genetics and clinical chemistry laboratory.
    Biomedica biochimica acta 02/1991; 50(1):3-10.
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    ABSTRACT: Proteasomes isolated and purified from rat muscle tissue and from the archaebacterium Thermoplasma acidophilum have a very similar size and shape, but the subunit composition is less complex in the archaebacterium as compared to the eukaryotic particle. The archaebacterial enzyme contains a catalytic site with chymotryptic specificity, which is inhibited by serine proteinase inhibitors and clearly differs from the eukaryotic particle which has a minimum of three catalytic sites for peptide bond hydrolysis of a yet undefined mechanism.
    Biomedica biochimica acta 02/1991; 50(4-6):465-9.
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    ABSTRACT: A class of cytosolic proteins has been identified that are degraded faster (have shorter half-lives) in human diploid fibroblasts deprived of serum. In RNase A, a model protein used for these studies, a pentapeptide comprising amino acids 7-11, Lys-Phe-Glu-Arg-Gln or KFERQ, is responsible for its enhanced degradation. The cytosolic proteins that are degraded faster during serum deprivation are recognized by an antiKFERQ antibody and, therefore, probably contain variations of the KFERQ motif. These cytosolic proteins are degraded in lysosomes. Transport into lysosomes in vitro is stimulated by ATP and the heat shock cognate protein of 73 kDa (hsc73).
    Biomedica biochimica acta 02/1991; 50(4-6):393-7.
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    ABSTRACT: The length of upper and lower incisors of Long-Evans hooded rats between gingiva and top were measured before and after small bilateral symmetric electrolytic lesions of the pontine reticular formation between the ventromedial parabrachial nucleus and the motor trigeminal nucleus. Ten days after lesions of this region 8.5 mm posterior to bregma the length of upper incisors was 13.4 +/- 0.7 mm compared with 6.1 +/- 0.2 mm before lesion (p less than 0.01) and the length of the lower incisors 17.1 +/- 0.4 mm compared with 10.2 +/- 0.2 mm before lesion (p less than 0.01). A second group with a smaller lesion of this region 9.0 mm posterior to bregma reached upper incisor length of 13.2 +/- 0.4 mm (p less than 0.05) and lower incisor length of 14.7 +/- 0.5 mm (p less than 0.05) at the 20th postoperative day. Sham-operated controls had no tooth growth acceleration.
    Biomedica biochimica acta 02/1991; 50(2):219-22.
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    ABSTRACT: Alterations in the erythrocyte rheology and the contents of activated free radical oxidation products (conjugated dienes, products of thiobarbituric acid and Schiff bases) in the acute phase of experimental thermic injury of the skin were studied. Erythrocyte flexibility reduction and erythrocyte aggregation increase correlated with elevated amounts of free radical oxidation products. Alpha-tocopherol avoided the accumulation of free radical oxidation products and improved both antioxidant defence and erythrocyte rheology. Thus we suppose that free radical oxidation products probably participate in the pathogenesis of erythrocyte rheology disturbances after thermic trauma.
    Biomedica biochimica acta 02/1991; 50(1):71-6.
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    ABSTRACT: Difference spectroscopy and enzyme kinetics were employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle with the azo-dye Procion Red HE-3B and two of its structural variants in order to follow the significance of the sulphonated terminal rings for the strength and specificity of binding. Procion Red HE-3B possesses a significantly higher affinity to LDH compared to the dye Cibacron Blue F3G-A, a well characterized pseudo-biospecific ligand of dehydrogenases. Moreover, Procion Red HE-3B showed competition towards the cofactor NAD+/NADH. The enzyme-dye complex is mainly stabilized by hydrophobic interactions, but other binding forces cannot be excluded. LDH possesses one dye-binding site per subunit. As a binding region the active center of LDH, preferentially the hydrophobic nicotinamide pocket is involved. Removal of the negatively charged sulphonic acid group from the terminal rings of Procion Red HE-3B decreases the affinity to LDH significantly but does not change the type of binding. Addition of an anilino group to the terminal rings of Procion Red HE-3B does not affect the affinity to the active site significantly but enables the binding on other sites with lower affinity in dependence on the dye concentration.
    Biomedica biochimica acta 02/1991; 50(12):1167-76.
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    ABSTRACT: In vitro protein folding is a spontaneous process that is driven by a small difference in Gibbs free energy between the native and unfolded states. The information required for correct folding should be entirely encoded in the amino acid sequence of the protein, although increasing evidence exist that proteins participate in cellular folding events. Isomerization of Xaa-Pro peptide bonds is thought to represent some slow steps of folding kinetics. This type of molecular reorganization have to be important in cellular folding due to the different isomeric states in proteins. Peptidyl-prolyl-cis/trans-isomerase (PPIase) catalyzes some, but not all, proline-limited slow folding reactions. On the other hand, the amino acid sequence of 17,8 kD PPIase from pig kidney is identical with cyclophilin (Cyp) that is the major cellular binding protein for the immunosuppressive drug cyclosporin A (CsA). The connection between enzyme catalyzed cis/trans isomerization, protein folding and immunosuppression is still unknown. PPIases of the cyclophilin type are found in most organisms and in various subcellular compartments. Recently a second family of PPIases has been discovered. These small proteins are structurally related to the cyclophilins; yet they bind with a high affinity to another immunosuppressive drug, the macrolide FK 506. Although it seems to be logical to ascribe the enzymatic activity of these proteins to a catalytic role in the folding of proteins within the cell other possibilities must also be considered and are discussed.
    Biomedica biochimica acta 02/1991; 50(10-11):S137-42.
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    ABSTRACT: The effect of dextran sulfate on the fusion of Sendai virus and erythrocyte ghosts was studied as a function of dextran sulfate concentration and pH of cell suspension solutions. In order to examine the interaction of dextran sulfate with Sendai virion and erythrocyte ghost surfaces, the turbidity of cell suspensions was also measured with respect to the same parameters as above. It was found that dextran sulfate inhibited the fusion of Sendai virus with erythrocyte ghosts. The lower the pH of the suspension solution was, the more effective was dextran sulfate in suppressing virion erythrocyte ghost fusion. Dextran sulfate of a higher molecular weight was slightly more effective in suppressing fusion than that of a lower molecular weight. From turbidity measurements of Sendai virus and erythrocyte ghost suspensions, it is likely that dextran sulfate interacts preferentially with Sendai virion surfaces and inhibits interaction between Sendai virions and erythrocyte ghosts.
    Biomedica biochimica acta 02/1991; 50(2):199-206.
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    ABSTRACT: The inactivation of soybean lipoxygenase-1 and of rabbit reticulocyte lipoxygenase by five selected acetylenic fatty acids was studied. In all cases the inactivation was time-consuming and depended on the concentration of the inactivator. The inactivation kinetics was measured and the data were fitted to a kinetic model based on the assumption of catalytic self-inactivation. The kinetic constants (Km-value and inactivation rate k2) calculated indicated that 7,10,13-eicosatrienoic acid was the most powerful inactivator for the soybean enzyme followed by 8,11,14-eicosatrienoic acid. The occurrence of an additional triple bond between C-4 and C-5 or between C-5 and C-6 strongly reduced the suicidal rate. With the reticulocyte enzyme, only small differences in the reactivities towards various acetylenic fatty acids have been observed.
    Biomedica biochimica acta 02/1991; 50(7):835-9.
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    ABSTRACT: In incubations with isolated rat liver mitochondria we studied the fuel properties of octanoate as medium-chain fatty acid and respiratory substrate and the extent of control exerted by adenine nucleotide translocase on mitochondrial respiration. While, compared with pyruvate, octanoate improved the hydrogen supply in the active state to be seen from a high reduction of the mitochondrial NAD(P) system and an increased delta psi, it also decreased the efficiency of energy transduction indicated by a low ADP/O ratio. Based on measurements of the dependence of respiration on the extramitochondrial ATP/ADP ratio, we conclude that a switch-over from pyruvate to fatty acid oxidation does not change the kinetic parameters which make respiration respond to the ATP/ADP ratio. It is shown that the decrease of the exchangeable intramitochondrial adenine nucleotide pool due to the activation of octanoate results in a decrease of the activity of the adenine nucleotide translocase and an increase of its flux control coefficient.
    Biomedica biochimica acta 02/1991; 50(7):841-9.
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    ABSTRACT: The role of water in mainly organic reaction mixtures for biocatalysis is best analysed in terms of the thermodynamic water activity. This determines water mass action effects on the equilibria of protease-catalysed peptide or ester synthesis. It can also be useful to predict the amount of water bound by the enzyme, and hence its catalytic activity, as other factors are changed.
    Biomedica biochimica acta 02/1991; 50(10-11):S61-6.
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    ABSTRACT: The effect of exogenous insulin on the maternal serum level of alpha-fetoprotein and fetal weight was studied. Insulin was given either as one subcutaneous injection on the 13th day of pregnancy (pulse treatment) or as subcutaneous injections each day throughout pregnancy (long-term treatment). Neither of the treatment procedures resulted in a significant alteration in the average fetal weight. However, pulse treatment on the 13th day of pregnancy resulted in elevated maternal serum levels of alpha-fetoprotein and in a greater number of fetuses and larger fetal mass per animal as examined on the 18th day of pregnancy. By contrast, long-term treatment with insulin from the first day of pregnancy resulted in a lower level of maternal serum alpha-fetoprotein and in significantly lower fetal mass per animal. It is concluded that insulin treatment as such, and especially the mode of treatment, plays a crucial role for fetal growth and synthesis of alpha-fetoprotein.
    Biomedica biochimica acta 02/1991; 50(7):901-6.
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    ABSTRACT: On the basis of amino acid sequences inferred from the genes encoding human neutrophil elastase and cathepsin G, it is likely that both are synthesized as precursors containing N- and C-terminal peptide extensions. We show that these extensions are removed about 90 min after onset of synthesis of these proteins in the U937 cell line. Removal of these extensions causes activation of the proteinases, and it is likely that the N-terminal extension of each enzyme serves as a zymogen activation peptide. Elastase and cathepsin G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Zymogen activation results from cleavage following a glutamic acid residue, a specificity opposite to most other serine proteinase zymogens. The specificity is likely to be shared, however, by neutrophil proteinase 3, rat mast cell proteinase II, and most members of the granzyme group of proteinases present in cytotoxic T-lymphocyte granules. The conservation in zymogen activation specificity between these leukocyte proteinase homologs is mirrored by the preservation of a discrete genomic organization. This suggests that most of the leukocyte serine proteinases evolved from a common ancestor distinct from the main branches of the chymotrypsinogen superfamily of serine proteinases.
    Biomedica biochimica acta 02/1991; 50(4-6):665-71.
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    ABSTRACT: The gene structures of rat cathepsins H and L have been determined. Cathepsin H gene spans more than 21.5 Kbp and comprises more than 12 exons. On the other hand, cathepsin L gene spans 8.5 Kbp and comprises 8 exons. In both genes, two intron insertion positions are conserved at the amino acid level. Both enzymes, therefore, diverged from a common ancestral gene. In the 5'-upstream region of the cathepsin L gene, no TATA box, one CAAT box and some SP-1 binding sites exist, common with other lysosomal enzyme genes. Furthermore, two AP-2 binding sites exist: a promoter under the control by the tumor promoter (TPA) and cAMP, and a cAMP response element (CRE). These results suggest that the cathepsin L gene expression is controlled by these factors.
    Biomedica biochimica acta 02/1991; 50(4-6):541-7.
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    ABSTRACT: The effect of various adrenergic agonists on autophagic sequestration--measured as the transfer of electroinjected [3H]raffinose from cytosol to vacuoles of the autophagic pathway--was investigated. Epinephrine and other agonists with alpha-effects inhibited sequestration through a specific alpha 1-adrenergic, i.e. prazosin-sensitive, mechanism. The beta-adrenergic agonist isoproterenol also inhibited sequestration, but by a non-beta-specific (propranolol-insensitive) mechanism. All sequestration-inhibitory agents suppressed overall autophagic-lysosomal proteolysis. The inhibitory action of the adrenergic agonists on protein metabolism was not specific to the autophagic pathway since protein synthesis was suppressed as well. However, intracellular levels of ATP were not adversely affected, ruling out the possibility that the agonists might be generally cytotoxic.
    Biomedica biochimica acta 02/1991; 50(4-6):383-7.
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    ABSTRACT: Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.
    Biomedica biochimica acta 02/1991; 50(10-11):S157-62.
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    ABSTRACT: 12 rabbits ("Deutscher Riese") were exposed to a hypoxic atmosphere (FiO2 = 0.05) for 3 h at their 1st day of life. At the 7th or 13th day of life, steel wire electrodes were chronically implanted into 3 leg muscles in order to record the electromyograms (EMG). The myographic activity was derived during free fall (height of drop = 0.5 m) until the age of 20 days. The quantitatively analysed EMG data were compared with corresponding data of undisturbed growing control animals. Using various EMG parameters, a quantitative separation of hypoxic animals from the controls was successfully done by a discriminant analysis, the animals being 8 and 20 days of age, respectively. It can be concluded that 1. already a single perinatal hypoxic load influences the supraspinal motor system permanently at least up to the age of 20 days, and that 2. this supraspinal influence on the investigated vestibulospinal reaction changes with increasing age of the rabbits.
    Biomedica biochimica acta 02/1991; 50(7):915-20.

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