Thomas A. Tedesco

Galactosemia: Symptomatic and asymptomatic homozygotes in one Negro sibship

The case histories of 2 Negro brothers are presented. Neither child hasgalactose-1-phosphate uridyl transferase activity in his circulating red cells, and thus each fulfills the laboratory criterion for the diagnosis of congenital galactosemia. One sibling demonstrated a classic galactosemic picture in infancy, while the other brother has never exhibited any signs or symptoms which could be attributed to galactosemia. Metabolic studies on the asymptomatic brother appear to place him in a subgroup of atypical “galactosemic patients,” as previously delineated by Segal and co-workers.5

Galactose-1-phosphate uridyltransferase and galactokinase activity in cultured human diploid fibroblasts and peripheral blood leukocytes: I. Analysis of transferase genotypes by the ratio of the activities of the two enzymes

The specific activities of galactokinase and galactose-1-phosphate uridyltransferase were determined in peripheral blood leukocytes directly after separation from whole blood, and in cultured skin fibroblasts at various times during the subculture growth period. Growth curves were obtained for fibroblasts based on three different parameters: direct cell counts, total protein, and total deoxyribonucleic acid (DNA) content. At the time in culture when the specific activity of both enzymes was maximal and least variable, the ratio of transferase to galactokinase correlated well with the transferase genotypes of the original tissue donors. Leukocyte transferase: galactokinase ratios gave a similar distribution pattern. Whereas transferase activity in both fibroblasts and leukocytes was similar, galactokinase was approximately three times as active in fibroblasts as in leukocytes. All fibrobast cell strains tested had similar galactokinase activity regardless of transferase genotype.The kinetic properties of fibroblast galactokinase were examined. Galactose-1-phosphate inhibits galactokinase activity in both normal and galactosemic cell strains, whereas other glycolytic intermediates have no effect. There was no detectable transferase activity in eight galactosemic (Gt(G)/Gt(G)) cell strains when transferase activity was maximal in cell strains of other transferase genotypes. Inhibitors responsible for the absence of transferase activity could not be demonstrated. In addition, transferase activity in galactosemic cell lysates was not observed in cells during logarithmic growth; measurable uridine diphosphate galactose (UDPgal) pyrophosphorylase activity was found in human diploid fibroblast cultures, as well as significant levels of endogenous uridine triphosphate (UTP) in lysates of fibroblast cultures.

Galactokinase: Evidence for a New Racial Polymorphism

Activities of galactokinase and galactose-1-phosphate uridyltransferase in red cells were assayed in a mixed racial population of 645 pregnant women. The distribution of individual transferase activities for black subjects was the same as that for whites. In contrast, the distributions of individual galactokinase activities differed significantly in blacks and whites, the mean for the black population being 30 percent lower than the mean for the white population. The same racial difference was found when red cell galactokinase activity was examined in males and in newborns. Because low-, intermediate-, and high-galactokinase activities appear to segregate within several black families, this observed difference suggests a new, racially determined enzyme polymorphism.

Linkage relationship between the genes for thymidine kinase and galactokinase in different primates

In this study we investigated the expression of primate galactokinase in somatic cell hybrids between a thymidine kinase-deficient mouse cell line and two different primate cell lines, one of which was derived from African green monkey kidney cells and the other from chimpanzee fibroblasts. All the African green monkey-mouse hybrid clones, selected in HAT medium, expressed monkey galactokinase activity and contained a monkey chromosome similar to a human E-group chromosome. When these clones were backselected in medium containing 5-bromodeoxyuridine, both this chromosome and the monkey galactokinase activity were lost. All the hybrid clones between mouse and chimpanzee cells, which were selected in HAT medium, contained the chimpanzee chromosome 17 and expressed chimpanzee galactokinase activity. These results indicate that the linkage relationship between galactokinase and thymidine kinase has been maintained in 3 divergent primate species—man, chimpanzee, and Old World monkey.

Assignment of the human gene for hexosaminidase B to chromosome 5.

Abstract Mouse-human somatic cell hybrids between different mouse and human cells were studied for the expression of human hexosaminidases A and B activities. The expression of human hexosaminidase B in the hybrids was found to segregate concordantly with the presence of the human chromosome 5. Mouse-human hybrid clones containing either the human chromosomes 5 and 7 only or the human chromosome 7 only were also included in this study. Expression of human hexosaminidase B activity was detected only in those clones containing human chromosome 5. These results indicate that the gene(s) for human hexosaminidase B is located on chromosome 5. No hexosaminidase A activity was detected in clones which contained either human chromosomes 5 and 7 or chromosome 7.

Inhibition of mammalian uridinediphosphoglucose 4-epimerase by the dithiothreitol-stimulated formation of NADH

Abstract 1. 1.|Human fibroblast lysates preincubated with dithiothreitol showed inhibition of UDPGlc 4-epimerase (EC 5.1.3.2) activity at endogenous levels of NAD + . This effect is probably due to NADH that is generated by the dithiothreitol stimulation of an NAD + -reducing system. Inhibition was reversed by the addition of (at least) 0.2 mM NAD + . A concentration-dependent inhibition of epimerase was also observed by NADH. 2. 2.|Several components of human fibroblasts and erythrocytes separable by gel electrophoresis are capable of NAD + but not of NADP + reduction in the absence of other added substrates. Another separate component is capable of NAD + and NADP + reduction only in the presence of dithiothreitol. These systems appear enzymatic in nature.

NONKETOTIC HYPERGLYCINEMIA (NKH): PERHAPS NOT SO RARE

Since 1976 four unrelated patients have presented with NKH. Only one was of consanguineous parents. One presented by 8 hours of life, the others within 3 days. Findings included myotonic jerks, lethargy, hypotonia and respiratory failure. Diagnostic laboratory data are:Other organic acid and amino acid metabolites were normal. Sodium benzoate and protein restriction were used in all four patients. Other therapy included strychnine, pyridoxine, phenobarbital and dilantin. Two died before 8 months, one before 2 years, one survives at 6 years with severe psychomotor delay. In order to have diagnosed 4 cases in 7 years in our catchment area we estimate the frequency of NKH to be about 1 in 60,000 births.

Studies on the molecular defect in galactosemia.

The galactose metabolic pathway and some of the consequences of deficient galactokinase or gal-1-P uridyltransferase activity have been discussed. The existence of CRM in transferase deficiency galactosemia is presented as evidence that this disease is the result of a structural gene mutation. The finding of both quantitative and qualitative variation in transferase CRM among different galactosemic patients argues that genetic heterogeneity exists within this group. Data supporting a Ping-Pong mechanism of action for human transferase reaction is proposed.

GENETICS: Normal, Duarte Variant, and galactosemic alleles code for immunologically identical gal-I-P uridyl transferase enzyme protein

Human galactose-l-phosphate uridyl transferase was purified from post-mortem liver to a preparation having a single band in polyacryamide gel electrophoresis. This preparation was used successfully to produce a rabbit antibody that precipitates transferase activity from solution and that forms a precipitin band in double immunodiffusion. Hemoglobulin-free erythrocyte preparations from homozygous normal (Gt+/Gt+), Durate Variant (GtD/GtD), and galactosemic (GtG/GtG) individuals show immunoprecipitin bands in double immunodiffusion against this antibody that are identical with that of the purified transferase preparation. The results indicate that the three alleles code for immunologically similar enzyme proteins suggesting that the functionally less active Duarte Variant and inactive galactosemic enzyme proteins have resulted from “point” mutations.