Biomedical subjects
Publications and source records attributed to D A Hopkinson.
Ten lectins have been used to detect glycoproteins, after SDS polyacrylamide gel electrophoresis and gel isoelectric focusing, in fibroblasts, red cell membranes, urine, and plasma of patients and obligate heterozygotes with cystic fibrosis. No disease specific changes were detected but considerable individual variation was observed, some of which was attributed to known genetic polymorphisms unrelated to cystic fibrosis.
Starch-gel electrophoresis was used to demonstrate two forms of glutathione S-transferase in human erythrocytes. Whereas considerable inter-individual differences in enzyme activity and electrophoretic patterns were detected, intra-individual differences were small.
A sensitive method for detecting isoenzymes of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in human liver biopsy specimens by simultaneous starch gel electrophoresis is described. All the currently recognised hepatic isoenzymes of ADH and ALDH which are concerned with ethanol metabolism can be identified. The method is suitable for amounts of liver biopsy material weighing as little as 2.5 mg and also allows spectrophotometric assay of ADH activity (at pH 8.8 and 11.0) and of ALDH.
A method has been developed for simultaneous analysis of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzymes in small (2.5 mg) liver biopsy cores by starch gel electrophoresis. All the currently recognized hepatic isoenzymes coded by ADH1, ADH2, ADH3 and ADH4 can be detected as can the five ALDH isoenzymes. Using this technique we have investigated the isoenzyme composition of liver samples from English and Chinese subjects and a group of chronic alcoholics. Pronounced racial differences in frequency of ADH2 and ALDH phenotypes were found--only 2 (4%) of English controls had the "atypical" ADH2 variant whereas this was present in 42 (84%) of Chinese subjects, and whereas all the English subjects had the rapidly migrating mitochondrial isoenzyme of ALDH, this was absent in 27 (54%) of Chinese. No differences in ADH or ALDH phenotype were seen in the chronic alcoholics, all of whom were of English origin, compared with the English controls, but there was a reduction in overall ALDH activity and particularly in the mitochondrial isoenzyme in those with cirrhosis. The reduction in ALDH activity is probably acquired; by limiting acetaldehyde oxidation it could be responsible for the rapid deterioration in liver function in patients who continue drinking excessively.
Two distinct isozymes (UMPH-1 and UMPH-2) which hydrolyse pyrimidine 5'-nucleotides have been identified in mouse, rat and Chinese hamster tissues. One of these, UMPH-1 appears to be specific for pyrimidine 5'-nucleotides, whereas the other, UMPH-2 has a broader substrate specificity. UMPH-1 and UMPH-2 show differences in their expression in different tissues. The evidence suggests that UMPH-1 and UMPH-2 are coded by distinct structural gene loci, Umph-1 and Umph-2. These isozymes have not been identified in man, and it is suggested that they may have overlapping electrophoretic mobility.
Electrophoretic and quantitative analysis of pyrimidine 5'-nucleotidase in human erythrocytes from healthy individuals and a patient deficient in pyrimidine 5'-nucleotidase, using a range of substrates, has shown that the patient has a marked deficiency with UMP, CMP and dCMP as substrates but near normal levels of activity with dUMP and dTMP as substrates. The observations suggest that two separate structural gene loci coding for distinct 5'-nucleotidases with similar electrophoretic mobility exist in man. The genetic determination of these enzymes seems therefore to be homologous with that found in rodents.
Analyses of DIA4 activity in cultured cells from a random population, a small family and human/rodent somatic cell hybrids support the view that the quantitative polymorphism of human DIA4 can be attributed to the segregation of a 'low activity' allele. (1) In a series of lymphoblastoid cell lines from 52 unrelated individuals, three lines were found which did not exhibit DIA4 after electrophoresis and showed low levels of NADPH diaphorase activity. The frequency (6%) of the DIA4 'absent' phenotype is close to that (4%) determined previously by analysis of post-mortem tissue. (2) Cultured fibroblasts from a small family in which the DIA 4 low-activity allele was apparently segregating were analysed. The NADPH diaphorase activity of the propositus (DIA4 'absent' phenotype) was very low (less than 6% of normal). He also exhibited very weak DIA4 isozymes of unusual electrophoretic mobility. The activity of both parents and sib was about half the normal level, suggesting that these individuals are heterozygous for the low-activity allele. (3) Analysis of a series of independent human/hamster hybrid clones, made using a human parent heterozygous for phosphoglycolate phosphatase (PGP) and DIA4, suggested that in this particular individual the DIA 4 low-activity allele segregates with the PGP 2 allele. However, there were 2 hybrids amongst a total of 16 which gave discordant results and these are to be the subject of further analysis by subcloning.
We report here a novel genetically determined polymorphism of a human urinary mucin which is demonstrable by the separation technique of SDS polyacrylamide gel electrophoresis, followed by detection with radio-iodinated lectins. The mucins are demonstrable using various lectins but the polymorphism is most easily recognized using peanut agglutinin and we therefore propose to designate this new genetic locus PUM (peanut-reactive urinary mucin). Four common alleles have been identified and an autosomal codominant mode of inheritance has been found in the families studied so far.
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1. A hydrophobic/phospholipid electrophoretic system has been devised which makes possible the analysis of human mitochondrial glycerol phosphate dehydrogenase (E.C. 1.1.99.5. GPDM). 2. GPDM has a wide tissue distribution in both adult and foetal life and is active in cultured lymphoblastoid cells and fibroblasts but is absent from red cells. 3. The solubilization procedure does not significantly alter the kinetic properties of the enzyme (Km alpha-glycerophosphate = 0.04-0.07 M, Km PMS = 0.19-0.35 mM) but the soluble form is less thermostable. 4. Comparisons of physicochemical characteristics, tissue distribution and coenzyme requirement point to a separate genetic determination and low level of evolutionary relatedness between GPDM and its cytosolic counterpart GPDS.
(1) There are very few clear examples among human enzymes of foetal isozymes which are the products of foetal specific gene loci. Earlier studies had pointed to the foetal brain esterase ESA7 as a probable example. (2) Detailed biochemical investigation of partially purified human adult brain ESA5 and the foetal esterase ESA7 has revealed a close resemblance in the biochemical properties of these two isozymes. In addition to similarities in substrate specificity and inhibition sensitivity the two esterases have the same molecular size (c. 57,000), are both relatively unstable at 37 degrees C and show decreased anodal electrophoretic mobility after storage at 20 degrees C. Furthermore there was suggestive evidence that ESA7 and ESA5 may be interconvertible. (3) A variant esterase isozyme pattern, which shows unusual features of both ESA7 and ESA5, was found in a survey of 120 foetal brains. This variant pattern is consistent with a monomeric structure for both esterases and points strongly to a common genetic determination.
1. The expression of soluble GPT (E.C. 2. 6. 1. 2) was analysed in seventeen independent rat hepatoma/human somatic cell hybrids and in forty-one subclones derived from two of these hybrids. 2. As judged by electrophoretic mobility, twelve hybrid clones expressed rat GPT activity only and three expressed strong rat and weak human GPT activity but no heteromeric isozymes. In the remaining two hybrids, only human GPT was demonstrable. 3. The segregation of GPT and marker enzymes in the primary hybrid cells and the subclones suggests that the human structural GPT locus is on chromosome 8. 4. The re-expression of rat GPT in segregating subclones derived from two primary hybrids which had extinguished this function, could not be correlated with presence or absence of any particular human chromosome(s).
1. The expression of twelve liver-specific enzymes was analysed in twenty-one independent rat hepatoma X human somatic cell hybrids, and in some cases up to forty-one subclones were also tested. 2. Seventeen hybrids continued to express most of the rat liver-specific enzymes and in some cases human isozymes of glutamate-pyruvate transaminase, alpha-glycerophosphate dehydrogenase, guanine deaminase, alcohol dehydrogenase and pyruvate kinase were clearly identified. 3. Analysis of the segregation of the human liver-specific enzymes in these hybrids led to the assignment of human GPT to chromosome 8 (previously reported, Kielty, Povey & Hopkinson, 1982) and suggests the assignment of human GPD1 to chromosome 12. 4. The expression of the various liver-specific enzymes in these hybrids appeared to be controlled by independent regulatory mechanisms. 5. Four unusual reverse segregant hybrids were also analysed, and in these no liver-specific enzyme activity was demonstrable.
The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.
A simple procedure for the preparation of soluble human succinate dehydrogenase is described. These preparations have proved suitable for analysis by zone electrophoresis, using a specific stain to detect activity after separation. In a survey of succinate dehydrogenase from various tissues and different individuals, no evidence for genetic heterogeneity due to the expression of either multiple loci or alternative alleles at the succinate dehydrogenase locus was found. However, epigenetic heterogeneity in both molecular size and charge was seen and various explanations for the occurrence of the isoenzymes are explored. Estimates of molecular size (93,300 +/- 9100) suggest that the smallest active unit of succinate dehydrogenase accounts for the major part of the solubilized activity. Kinetic studies have shown that the apparent Km values for succinate (0.9 mM) and PMS (0.4 mM) are comparable to those previously described for the beef heart enzyme, and these parameters were not significantly altered when the enzyme was removed from the membrane milieu. However a marked non-succinate-dependent activation of the membrane-associated enzyme at 38 C is apparently lost on solubilization, and this observation may have some bearing on earlier reports of an apparent decrease in Vmax on solubilization of succinate dehydrogenase.
Adenosine deaminase activity has been measured in red cells from individuals of known ADA phenotype (ADA 1, ADA 2-1, ADA 3-1, ADA 3-2) using adenosine and 2'-deoxyadenosine as substrates. No significant differences were observed among the phenotypes in their relative deaminase activity with the two substrates. However, evidence suggests the occurrence of an uncommon allele designated ADA1w determining low levels of ADA activity. The deaminase activities of the phenotypes were in the order ADA 1 greater than ADA 2-1 greater than ADA 3-1 greater than ADA 3-2 with both substrates. The relative activities of the alleles were estimated to be: ADA1 100%, ADA2 89%, ADA3 28% and ADA1w 67% with adenosine, and ADA1 100%, ADA2 87%, ADA3 39% and ADA1w 66% with 2'-deoxyadenosine. The Michaelis constants for adenosine and 2'-deoxyadenosine were determined for the different phenotypes. There were no significant differences in these values among the phenotypes.
Phosphoglycolate phosphatase (PGP) exhibits a wide range of activities in normal human red cells. Analysis of blood from 57 individuals of known PGP phenotype revealed no correlation between enzyme activity, electrophoretic phenotype or 2,3-DPG concentration. Neither was there evidence of variation in Km, heat stability, pH optimum or molecular size among the various electrophoretic phenotypes. However, human red cell PGP exhibits high (0.24 mM) and low (0.05 mM) Km values for phosphoglycolate irrespective of phenotype and this requires further analysis.
Nineteen enzymes showing highest activity in liver were examined in human and rodent tissues and cultured cells using starch-gel electrophoresis. The rat hepatoma line Faza 967 strongly expressed 13 of these enzymes. A series of somatic cell hybrids, constructed between Faza and cells of non-hepatic origin derived from man or from Chinese hamster, were examined for expression of these enzymes. Some of the human/rat hybrids continued to produce rat liver-specific enzymes, and the human forms of the enzymes glutamate-pyruvate transaminase, alpha-glycerophosphate dehydrogenase, alcohol dehydrogenase and pyruvate kinase L were reexpressed in a few cases.