Bohdan Bakay

Hypoxanthine-guanine phosphoribosyltransferase variants: Correlation of clinical phenotype with enzyme activity

A deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is associated with a varying clinical picture which may include hyperuricaemia, neurological abnormalities and bizarre self-multilating behaviour. Due to technical problems with the usualin vitro enzyme assays, it has not been possible to establish a correlation between the degree of the enzyme deficiency and the severity of the clinical manifestations. In this study, the HGPRT activity of 12 patients with various clinical features was measured by quantitative analysis of the incorporation of radioactive precursors into purine compounds in intact fibroblasts. The results demonstrate that a correlation between the severity of the clinical symptoms and the degree of the enzyme deficiency as measured in intact fibroblasts does in fact exist.

The separation of adenine and hypoxanthine-guanine phosphoribosyl transferases isoenzymes by disc gel electrophoresis

Hypoxanthine-guanine (HGPRT; E.C. 2.4.2.8) and adenine (APRT; E.C. 2.4.2.7) phosphoribosyl transferases were studied by disc electrophoresis on polyacrylamide gel. The positions of the isoenzymes were detected by radiochemical enzyme assay. The nucleotide products of the reactions were precipitated in the gel with lanthanum chloride. APRT was found to migrate slightly less rapidly than albumin and produced a single narrow symmetrical peak of activity. HGPRT migrated 25–50% more slowly than albumin and produced a broad zone of activity consisting of four unequal peaks. The APRT enzyme of Rhesus monkey liver and the HGPRT enzyme of sheep erythrocytes migrated notably slower than the corresponding human enzymes. An isoenzyme of APRT was detected in human erythrocytes which migrated more rapidly than that of most individuals. In all instances, the adenine was utilized by one electrophoretic component and hypoxanthine and guanine by another. Furthermore, the components which utilized hypoxanthine and guanine were inseparable. The sensitivity of the assay made it possible to assess the electrophoretic and enzymatic characteristics of HGPRT isoenzymes on aliquots of hemolysates capable of producing 0.5 picomoles of IMP per minute. In human erythrocytes with normal enzyme content, this amount of activity is present in approximately 50 nanoliters of cells.

Analysis of radioactive and nonradioactive purine bases, nucleosides, and nucleotides by high-speed chromatography on a single column.

Abstract The concentrations of radioactive and nonradioactive purine bases, purine nucleosides, purine mono-, di-, and trinucleotides in acid extracts of fibroblasts were determined by anion-exchange column chromatography. The concentrations of nonradioactive components were determined by computerized integration of the signal from a double-beam uv-detector. The radioactive metabolites were quantitated by high-efficiency, continuous liquid scintillation counting, employing a discrete sample transport system.

Detection of heterozygous carriers of the Lesch-Nyhan syndrome by electrophoresis of hair root lysates

Mosaicism has been demonstrated for the X-linked enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) in heterozygous carriers of the Lesch-Nyhan syndrome by examining single hair follicles for the activity of HGPRT and adenine phosphoribosyl transferase (APRT) using polyacrylamide gel electrophoresis. The method is useful in the detection of the carrier state and has advantages over methods previously published.

Electrophoretic properties of hypoxanthine-guanine phosphoribosyl transferase in erythrocytes of subjects with Lesch-Nyhan syndrome.

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C. 2.4.2.8) has been studied in erythrocytes of patients with Lesch-Nyhan syndrome by polyacrylamide gel electrophoresis. The location of this enzyme in gel was determined by radiochemical assay. Inosine monophosphate (the reaction product of HGPRT with radioactive hypoxanthine and 5-phosphorylribose-1-pyrophosphate) was precipitated in the gel at the site of its formation with lanthanum chloride. The zone containing radioactive inosine monophosphate was located by continuous monitoring of mechanically fractionated gels in a scintillation spectrometer. The sensitivity of this method has permitted the detection of the very low HGPRT activity in the electropherograms of hemolysates of patients with Lesch-Nyhan syndrome. Among six patients, four had a mutant enzyme which migrated 15% faster than the normal; the other two had a mutant enzyme with about 12% faster migration. These mutants were designated HGPRT-LN and HGPRT-LN slow, respectively. These observations indicate that the mutant gene on the X chromosome codes for a protein of altered structure.

Isoenzymes of hypoxanthine-guanine-phosphoribosyl transferase in a family with partial deficiency of the enzyme

The isoenzymes of hypoxanthine-guanine-phosphoribosyl transferase (HGPRT; E. C. 2.4.2.8) were studied by polyacrylamide gel disc electrophoresis in the erythrocytes of a family in which there was a partial deficiency of this X-linked enzyme. Hyperuricemic males, in whom HGPRT activity was 4% of normal, were found to have a variant enzyme which had altered kinetic and electrophoretic properties. In acrylamide gel, this variant migrated about 15% faster than the normal enzyme, and its Km for hypoxanthine was twice that of the normal. The sister of two patients had 34% of normal activity in her erythrocytes and was thought to be a heterozygote. Electrophoresis of her hemolysate yielded profiles in which there were two zones of HGPRT activity. The more slowly migrating isoenzyme behaved electrophoretically like the normal isoenzyme. The faster-migrating isoenzyme had a mobility identical to that of the variant enzyme found in hemolysates from her hyperuricemic siblings. However, in her profile the activity of the variant enzyme was three times greater than that of the HGPRT found in the boys. This increased activity appears to be due to an interaction of the variant enzyme with the normal enzyme. Electrophoresis of a mixture of normal enzyme and the variant from a hyperuricemic male yielded a profile similar to that observed in this girl and a dramatic increase in the amount of activity in the variant zone.

On the component proteins of calf thymus nucleoprotein.

Abstract By a study of experimental variables, a standardized procedure for the preparation of calf thymus nucleoprotein of essentially constant phosphorus and sulfur content has been developed. The nucleic acid-free protein has been isolated quantitatively by a modified Sevag-Mirsky procedure. The protein has been quantitatively separated into histone and residual protein by treatment with HCl at pH 2.5. The histone precipitated as a single substance, but on ultracentrifugation showed two components. These have been separated and partly characterized. One (“fast”), representing 63% of the histone and yielding molecular weight estimates of 130,000–140,000, contains 0.23% tryptophan, 0.67% cystine, and 2.4% methionine. The other component (37%, “slow”), of estimated molecular weight of 11,000–16,000, contains0.82% methionine, and is free of tryptophan and cystine.

Detection of radioactive components in polyacrylamide gel disc electropherograms by automated mechanical fractionation

An accurate, expedient method has been developed for the automated assay of radioactivity in polyacrylamide gels. In this method gels are fractionated following disc gel electrophoresis with a mechanical fractionator, mixed with an eluent carrier and then with scintillation solution, and passed through a flow cell. The flow cell can be monitored either by a single-channel scintillation spectrometer equipped with a rate meter and a single-pen chart recorder, or by a multichannel spectrometer. Analysis of gels 10 cm in length requires about 130 ml of scintillation solution and is completed in about 45 min. Assays of high- and low-concentration gels containing profiles of 3H- and 14C-labeled nucleotides (the products of enzymic reactions of adenine and hypoxanthine-quanine phosphoribosyltransferases) have shown that this method is accurate, highly discriminating, sensitive, and substantially more expedient than manual methods of slicing and digestion.

Heterogeneity of hypoxanthine guanine phosphoribosyl transferase from human erythrocytes

Abstract Hypoxanthine guanine phosphoribosyl transferase (E.C.: 2.4.2.8) has been purified 4000- to 4500-fold from normal human erythrocytes by three different schemes of protein fractionation. In one scheme, the enzyme was separated by preparative polyacrylamide gel electrophoresis in an LKB Uniphor system and purified by affinity column chromatography employing Sepharose/phosphoribosyl/pyrophosphate. In the second, the enzyme was isolated by isotachophoresis in the presence of Amphiline carrier ampholytes employing a Tris/phosphate/β-alanine ion system. The enzyme was then purified by isotachophoresis in the presence of carrier ampholytes using a Tris/acetate/glycine ion system. The hypoxanthine guanine phosphoribosyl transferase purified by affinity chromatography and isotachophoresis consisted, on immunoelectrophoresis, mainly of one component and had less than 5% impurities. When subjected to analytical polyacrylamide gel electrophoresis, such preparations were resolved into four isoenzymes. In the third scheme, the enzyme was isolated by isoelectric focusing. In this system, the enzyme was also resolved into four isoenzymes. Their isoelectric points were: 5.47, 5.63, 5.74, and 5.84. When subjected to analytical polyacrylamide gel electrophoresis each isoenzyme migrated at a different rate. In sodium dodecyl sulfate gel electrophoresis each isoenzyme yielded one major and one minor band. Protein appearing in the major and minor bands migrated at rates consistent with a molecular size of 33,500 and 26,500, respectively.

A novel method of sample transport and its application for continuous detection of radioactivity in the effluent of the high speed amino acid analyzer.

Abstract A method for detection and transport of samples in small bore conduits utilizing a semisolid resilient gel spacer is described. In combination with a hollow tube flow cell, this method of transport permitted monitoring of the effluents from a high pressure-high speed amino acid analyzer for radioactivity without loss of resolution at efficiencies that are comparable to those obtained in common counting vials.