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Identification of Galactose6Phosphate in Galactosæmic Erythrocytes
Abstract
GALACTOSÆMIA is a hereditary metabolic disease characterized by the absence of galactose-1-phosphate uridyl transferase1 with accumulation of galactose-1-phosphate2. Yet, in the liver, small quantities of galactose-1-phosphate may be utilized and incorporated into uridine diphosphate galactose by the pyrophosphorylase3. In vitro studies with crystalline, muscle phosphoglucomutase have shown that galactose-1-phosphate can be converted to galactose-6-phosphate4. This communication presents evidence of galactose-6-phosphate during the utilization of galactose by galactosæmic erythrocytes.
... Actually four alternative pathways for galactose utilization by galactosemic cells have been proposed. They involve the conversion of: (1) galactose-I-phosphate (gal-I-P) to uridine diphosphategalactose (UDP-gal) by a pyrophosphorylase (Isselbacher, 1957); (2) galactose toD-xylulose (Cuatrescasas and Segal, 1966); (3) galactose to 6-phosphogalactonate (6-P-gal) (Inouye et al., 1962); and (4) galactose to galactonolacte,ne and galactitol (Kalckar et a1., 1959;Segal et al., 1965). It has also. ...
Hexose 6-phosphate dehydrogenase, a microsomal enzyme form rat liver, has been shown to reduce NAD⁺ or NADP⁺ in the presence of galactose-6-P or glucose-6-P. The reaction product of hexose 6-phosphate dehydrogenase and galactose-6-P, uniformly labeled with ¹⁴C was isolated by Dowex 1-chloride column chromatography. It readily formed lactone on boiling with 1 n HCl and was identified as 6-phosphogalactonic acid by paper chromatography. Incubation of liver fractions with 6-phosphogalactonic acid-1-¹⁴C resulted in formation of only minimal amounts of ¹⁴CO2, quantities that could readily have come from trace contaminants of 6-phosphogalactonic acid-1-¹⁴C.
The product of glucose-6-P oxidation by hexose 6-phosphate dehydrogenase and NAD⁺ provided substrate for the reduction of NADP⁺ in the presence of 6-phosphogluconic acid dehydrogenase, and was hence identified as 6-phosphogluconic acid.
Incubation of uniformly labeled galactose-¹⁴C with rat liver supernatant fractions failed to disclose the formation of detectable quantities of galactonic acid. Thus, the existence of a "galactose dehydrogenase" in rat liver could not be confirmed.
Biochemical disease involves a departure from the normal in some body component, e.g. an enzyme, a structural protein or a membrane transport mechanism. Some biochemical diseases are almost entirely environmental in origin, e.g. lead encephalopathy, but at present attention is largely directed to genetically determined conditions-the inborn errors of metabolism and other molecular diseases. In some cases it is the interaction between genotype and external environment which brings about the signs and symptoms, in others, such as the gangliosidoses, no modification of the external environment can appreciably alter the course of the disease.
Galactosemia is a serious disease that leads to death in infancy in many cases. Almost all the untreated survivors suffer from mental retardation, cataracts, and varying degrees of damage to liver and kidneys. This chapter describes the widespread and variable clinical features of galactosemia, and provides an account of experimental galactosemia in animals. The biochemical studies such as normal metabolism of galactose, the nature of the enzyme defect in galactosemia, the biochemical consequences of this defect, and its correlation with the clinical aspects are also reviewed. Galactosemia cannot be diagnosed without aid from the laboratory. Thus, the chapter provides an overview of laboratory procedures for the diagnosis of galactosemia in the newborn and later in life, for the monitoring of treatment and for the detection of carriers. Like the other inborn errors of metabolism, galactosemia is a genetically determined disease. The direct assay of enzymatic activity in heterozygotes for galactosemia is a major contribution of biochemistry to the study of human genetics. The chapter discusses dietary treatment of galactosemia and the different diets proposed for the galactosemic patients. The clinical and biochemical features of familial galactose intolerance and congenital hepatic and renal dysfunction resemble the features of galactosemia and thus, make the diagnosis of galactosemia difficult.
Most galactose in the diet is in the form of the disaccharide lactose, which is synthesized in the mammary gland, mainly from glucose. Lactose is the primary carbohydrate source for nursing mammals and it provides about 40% of the energy in human milk. Why lactose evolved as the unique carbohydrate of milk is unclear, especially since most individuals can meet their galactose needs by biosynthesis from glucose. The simultaneous occurrence of calcium and lactose in milk may be of evolutionary significance. Lactose, in contrast to other saccharides, appears to enhance the absorption of calcium1,2. In man, calcium absorption may be associated with the hydrolysis of lactose3.
The human erythrocyte offers an opportunity for investigation of galactose metabolism because the complete normal pathway exists in this accessible tissue. The erythrocyte has proven to be useful not only in identification of the heterozygous and homozygous states in galactosemia and in the galactokinase defect, but also in the study of variants. It is recognized, however, that the erythrocyte is a special type of cell and that findings from its study should not be extended to other tissues without reservation. With respect to the erythrocyte itself, there still is much to be learned concerning intracellular kinetics under different conditions and the nature of the enzymes involved.
D-Galactose 6-phosphate as synthesized by direct phosphorylation of D-galactose with polyphosphoric acid is contaminated with two of its positional isomers. These were separated from D-galactose 6-phosphate and from each other, and identified as D-galactose 3- and 5-phosphate by enzymic, chromatographic, and mass-spectral analysis. The previous misidentification of these isomers as furanose forms of D-galactose 6-phosphate has led to erroneous reports concerning the anomeric distribution of D-galactose 6-phosphate. The anomeric distribution of D-galactose 6-phosphate in a purified preparation was determined by gas-liquid chromatography and 13C-n.m.r. spectroscopy to be 32% α-pyranose, 64% β-pyranose, and no more than 4% furanose anomers.
GALACTOSE-6-phosphate (gal-6-P) has been shown to be produced from galactose-1-phosphate (gal-l-P) in the presence of phosphoglucomutase1, demonstrated in eryth-rocytes of patients with galactosæmia2, and oxidized in the presence of purified glucose-6-phosphate dehydrogenase (G-6-PD) and NADP as indicated by changes in absorbance at 340 mµ (ref. 3). This communication shows that G-6-PD purified from either yeast or human erythrocytes in the presence of NADP converts gal-6-P to 6-phosphogalactonic acid (6-P-gal A).
The present paper reports on the enzyme studies performed on 421 individuals in twenty-seven kindreds which include twenty-nine sibships containing galactosemic individuals.
By fitting normal curves to a group of known heterozygotes (parents) and normal controls, it was possible to establish exclusion limits for each group. The analysis indicates that most heterozygotes can be classified correctly on the basis of enzyme studies. The identification of heterozygotes confirms the previous suggestion that galactosemia is due to an autosomal recessive gene.
In the galactosemic sibships, there was an excess of males over that usually seen in the white population at birth. This effect appeared to be present among both affected and unaffected children and was independent of birth order and parental age.
This paper summarizes current thinking on the methods for the assay of galactose-1-phosphate uridyl transferase and how they are being used to detect clinical variants of galactosemia.
The transport and phosphorylation of 2-deoxy-D-[3H]galactose in rabbit renal cortical cells was studied. 1. The uptake of 2-deoxy-galactose by cortical slices is associated with an appearance of both free and phosphorylated sugar in the cells. At 1 mM external sugar the cells establish a steady-state gradient of free 2-deoxy-galactose of 3.97 +/- 0.15 (23 animals). 2. The acid-labile sugar phosphate accumulated in the tissue has been identified by a combination of paper and radio-chromatography, as well as on the basis of some of its chemical properties, as 2-deoxy-D-galactose 1-phosphate. Ice-cold trichloroacetic acid produces a decomposition of this compound. 3. Increasing external pH (6-8) brings about a decrease in the steady-state levels of both free and phosphorylated sugar in slices. On the other hand, increasing pH activates the phosphorylation of 2-deoxy-D-galactose by a crude kinase in a tissue extract. 4. Sugar phosphate accumulated in the cells is dephosphorylated by the action of a Zn2+ -activated phosphatase. 5. The efflux of 2-deoxy-D-galactose from the cells is rather slow compared with that found for D-galactose. The efflux is associated with some dephosphorylation of cellular sugar phosphate, and some loss of 2-deoxy-galactose phosphate into the wash-out medium takes place. 6. An inhibition analysis of the uptake of 2-deoxy-D-galactose by the slices indicates that the transport site is shared by D-galactose. The following points of interaction between the sugar molecule and the carrier are identified: C1-OH, C3-OH and C4-OH (both axial) and C6-OH. A (pyranose) ring structure is also essential. A close packing between the substrate and the carrier in the vicinity of C2 is indicated. 7. The data suggest that the above transport system is localized predominantly at the antiluminal (basolateral) face of the renal tubular cells. While the detailed mechanism of the actual transport step (i.e. active transport of the free sugar, or by the action of a phosphotransferase) is still unclear, the data present evidence that both galactokinase and a Zn2+ -activated phosphatase participate in the maintenance of an intracellular steady state of the transported sugar.
A new enzyme, galactose-6-phosphate dehydrogenase has been purified about 50-fold from goat liver. The enzyme can be distinguished from the nonspecific hexose-6-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase by its high substrate specificity and absolute pyridine nucleotide requirement. In contrast to the hexose-6-phosphate dehydrogenase, this enzyme is located exclusively in the cytoplasmic fraction of the cell. The enzyme is a metalloprotein and is highly sensitive to mercurials. The product of the reaction is possibly a ketoaldose, phosphorylated at the primary alcoholic group.
UDPglucose-4-epimerase (EC 5.1.3.2) is shown to exist in two distinct forms in goat and beef liver. The two forms can be distinguished easily by their interaction with sugar phosphates and with substrate. The minor form comprising about 20% of the total activity, is activated by both and Gal-6-P and is partially inhibited by . None of these sugars have any effect on the major form except Gal-6-P, which inhibits its activity partially only in the case of goat liver. The major form, however, undergoes inhibition by substrate at higher concentrations. The two forms behave similarly in their requirement for NAD for activity and in their inhibition by UMP. The induced epimerase in yeast fails to show the existence of these two forms.
Leloir pathway enzyme activities of adult rat heart were present in amounts 8, 38, and 23% that of adult rat liver for galactokinase, galactose 1-phosphate uridyl transferase, and UDP-galactose 4′-epimerase, respectively. The corresponding values for the adult rat brain enzymes relative to liver were 27, 11, and 120%, respectively. Michaelis constants for the three enzymes were comparable in heart, brain, and liver of the adult rat. While diminished galactokinase activity correlated well with the established limited ability of brain and heart to metabolize galactose, the low uridyl transferase activity of adult rat brain could also contribute to its restricted galactose utilization.Rat heart and brain, but not liver, soluble enzyme preparations produced in addition to galactose 1-phosphate, a metabolite tentatively identified as galactose 6-phosphate when incubated with high concentrations of d-galactose (30 mm).Galactose 6-phosphate was further identified in the brains of galactose-intoxicated chicks and rat hearts perfused with d-galactose by spectrophotometry and analysis by combined gas-liquid chromatography-mass spectrometry. Tissue concentrations of galactose 6-phosphate were compared to selected glycolytic and galactose metabolites.
Cultures of human galactosemic fibroblasts without detectable transferase activity were able to convert [1-14C]galactose to 14CO2 to the same extent as normal cells, but did so at a significantly slower rate. The utilization of galactose in both normal
and galactosemic cells was strongly inhibited by glucose at physiologic concentrations.
A fluorometric method for the assay of galactose-1-phosphate in erythrocytes is described, based on the action of alkaline phosphatase and galactose dehydrogenase. With this method galactose-6-phosphate is also measured, but not UDPGalactose (uridinediphosphogalactose). The findings in control subjects, 1 patient with the classical form of hereditary galactosemia (deficiency of galactose-1-phosphate uridyltransferase), and 1 patient with hereditary galactokinase deficiency are described. The method is intended for control of the effectiveness of dietary treatment in patients with the classical form of hereditary galactosemia, but it can also be used for the differential diagnosis between this disease and galactokinase deficiency.
The effectiveness of galactose as a substrate for methaemoglobin reduction in human red cells has been investigated. The rate of methaemoglobin reduction has been found to depend on the concentration of galactose up to levels of 0.5 M. Methylene blue, which markedly accelerated methaemoglobin reduction when glucose was a substrate, only minimally accelerated methaemoglobin reduction with a galactose substrate. Pre-incubation with high concentrations of galactose did not inhibit methaemoglobin reduction in the presence of glucose and methylene blue. No inhibition of G-6-PD activity was observed with very high concentrations of Gal-1-P, glucose-1-P, or galactose, even at low concentrations of G-6-P. Several possibilities are presented to explain the lack of stimulation of methaemoglobin reduction by methylene blue when galactose is the substrate: 1) inhibition of methylene blue-stimulated methaemoglobin reduction by galactose or its metabolites; 2) inability of the red cell to metabolize very low concentrations of glucose-6-phosphate by way of the hexose monophosphate pathway; and 3) that β-glucose-6-phosphate is not an important product of galactose metabolism. In the latter case, which appears the most likely, a previously undescribed pathway of carbohydrate metabolism must exist in the human erythrocyte. It is suggested that this pathway may represent the direct conversion of α G-6-P to fructose-6-phosphate, by-passing the hexose monophosphate pathway, which requires β G-6-P as substrate.
Starch-gel electrophoresis of extracts of human liver revealed the presence of a new hexose-6-phosphate dehydrogenase that was slower-moving at pH 8.6 than the sex-linked glucose-6-phosphate dehydrogenase. When the gel plate was stained, galactose-6-phos phate being used as a substrate, this enzyme band stained intensely, but the sex-linked glucose-6-phosphate dehydro genase failed to stain. This new human enzyme may well be homologous with the autosomally inherited glucose-6-phosphate dehydrogenase of the deer mouse (Peromyscus maniculatus), re ported by Shaw and Barto.
La phosphoglucomutase du muscle de lapin convertit l'acide α-galactose-1-phosphorique en une substance acidostable qui est vraisemblablement l'acide galactose-6-phosphorique.
The reduction of sugars to the corresponding alcohols is shown to be smoothly effected by sodium borohydride in aqueous solution.
Galactosemia seems to furnish an example of a congenital human metabolic disease in which a specific enzyme is missing. The enzyme which catalyzes the exchange of α-galactose-I-phosphate with uridinediphospho-glucose, forming α-glucose-I-phosphate and uridinediphospho-galactose is absent in blood from galactosemic subjects. It is known that this enzymic exchange is an important reaction by which administered galactose is used in general carbohydrate metabolism. Several of the metabolic manifestations of the disease might readily be explained on the basis of this enzymatic defect.