Galactose-l-phosphate

Enzymes, measured in clinical laboratories, for which kits are available include y-glutamyl transferase (GGT), alanine transferase [9000-86-6] (ALT), aldolase, a-amylase [9000-90-2] aspartate aminotransferase [9000-97-9], creatine kinase and its isoenzymes, galactose-l-phosphate uridyl transferase, Hpase, malate dehydrogenase [9001 -64-3], 5 -nucleotidase, phosphohexose isomerase, and pymvate kinase [9001-59-6]. One example is the measurement of aspartate aminotransferase, where the reaction is followed by monitoring the loss of NADH ... 

FIGURE 19.34 The galactose-l-phosphate uridylyltransferase reaction involves a ping-pong kinetic mechanism. 

Schwarz and Golberg (S8) found that the lenses of galactose-fed rats contained 10 times the normal concentration of galactose-l-phosphate and Lerman (L4) suggested that this inhibited the normal enzymic activity of the lens. 

Reaction (1) is irreversible the enzyme galactokinase has been found in mammalian liver, brain, and erythrocytes, as well as in certain yeasts and other microorganisms. UDPGal, the main product of reaction (2), is the intermediate by means of which the body incorporates galactose into cerebrosides and, probably, other galactolipids, mucopolysaccharides, and lactose galactose-l-phosphate uridyl transferase occurs in the liver, to a lesser extent in red cells, and probably in other tissues. 

Gal-l-P + UDPG = G-l-P + UDPGal Galactose-l-phosphate uridyl transferase... 

The galactose-l-phosphate uridyl transferase pathway is by far the most important in galactose metabolism it accounts for about 5/6 of the total galactose metabolized in mammals. It is less active in the newborn than in the adult, e.g., the liver of newborn rats has only 1/5 the adult amount of galactose-l-phosphate uridyl transferase per milligram of protein (13). 

A similar reaction had previously been known to occur in Saccharomyces fragilis (K8) and in bean seedlings (N3). This is an alternative route to UDPGal, but the activity of the enzyme in newborn liver is very low even in the adult it is only one-sixth that of galactose-l-phosphate uridyl transferase (13), as shown in Table 2. 

Schwarz et al. (S10) showed that the erythrocytes of galactosemic subjects accumulated large amounts of galactose-l-phosphate, both in vivo and in vitro. This indicated that the metabolic block in galactosemia occurred at reaction (2) or reaction (3). Kalckar et al. (K7) and Isselbacher et al. (19) later showed that normal erythrocytes contained all 4 enzymes needed for reactions (1), (2), (3), and (4), and that in galactosemia there was virtually complete absence of galactose-l-phos-... 

All the varied biochemical and clinical features of galactosemia are, beyond any reasonable doubt, caused by the absence of this one enzyme, galactose-l-phosphate uridyl transferase. Yet it is not possible, at present, to identify all the steps by which the different effects are brought about. 

About 10% of the acid-soluble phosphorus of the red blood cells in galactosemia is accounted for by galactose-l-phosphate. Since all this is derived from adenosine triphosphate by reaction (1), it represents the tying up in a metabolically useless form of a high proportion of the high-energy phosphate of the erythrocyte. Untreated galactosemics have... 

Glucose-6-phosphatase is another enzyme competitively inhibited by galactose-l-phosphate (15). The inhibition of hepatic glucose-6-phos-phatase is probably important in relation to hypoglycemia. 

It seems established that galactose-l-phosphate is an enzyme poison, blocking the cell s most important source of energy. The failure to thrive characteristic of galactosemia is thus explained the less severely affected infants were possibly exposed to lower concentrations of galactose-l-phosphate, either because of their diet or because of some peculiarity of constitution. 

It was demonstrated by Schwarz et al. (S10) that red blood cells of a galactosemic individual, on incubation with galactose either in vivo or in vitro, accumulate galactose-l-phosphate. This has been made the basis of a diagnostic test for galactosemia (S6, S9). Red blood cells are... 

A better method has been described by Schwarz (S6). Washed red blood cells are lysed, precipitated with trichloroacetic acid below 0°C and the supernatant quickly neutralized. Speed and low temperatures are necessary to prevent hydrolysis of galactose-l-phosphate which is very sensitive to acid. Barium acetate and ethanol are added, and the precipitated barium salt of galactose-l-phosphate washed with 80% ethanol. The barium salt is then hydrolyzed by heating with dilute hydrochloric acid, acid and salts removed with mixed ion-exchange resins, and the galactose estimated by paper chromatography as described above. It is probably better to avoid the Amberlite MB-1 resin used by Schwarz and, instead, to use a weak base resin mixture, such as Amberlite MB-4. Recovery of added galactose-l-phosphate should be determined simultaneously. 

An enzymatic method (Kll) of estimating galactose-l-phosphate in red blood cells is simpler and less time-consuming than the paper chromatographic method, though it requires more expensive reagents and apparatus. This method closely resembles methods of assaying enzyme activity and it is, therefore, described with them in Section 4.4.1. 

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