Motoshi Kitamura

Occurrence of persistent elevated levels of serum glutamic oxaloacetic transaminase as a result of enzyme-immunoglobulin complex formation with a report of two cases

SummaryTwo cases of macro-molecular GOT (glutamic oxaloacetic transaminase) were encountered recently. Both were middle-aged women, showing no abnormality in laboratory test results including those for GPT (glutamic pyruvic transaminase), except that abnormally high values (ca. 200 Karmen unit) were obtained for GOT. Clinical examinations and tests on the liver, heart, skeletal muscle and other organs were negative. By column chromatography, immunoelectrophoresis and other procedures, this macro-molecular GOT was shown to be an anzyme immunoglobulin complex with a molecular weight of about 250,000 formed by the binding of the GOT of the cytoplasmic fraction to Ig-G-Kappa.

Molecular characterization of genetic mutations in human lactate dehydrogenase (LDH) B (H) variant

SummaryWe have previously detected a single base substitution of G by A at the Arg codon CGC in exon 4 of the mutant lactate dehydrogenase (LDH) gene, an unstable LDH-B variant (case 1). Here, we use the polymerase chain reaction (PCR) to amplify genomic DNA of two cases (the original case 1 and a new patient, case 2). We were able to confirm that case 1 is homozygous for the mutation, causing a replacement of the conserved Arg by His at residue 173. The resulting LDH-B variant subunit is unstable in vivo. Whereas the mutation in exon 4 was not observed in case 2, a different single base substitution of A by C was detected at the Ser codon AGT in exon 3. This mutation causes a replacement of the conserved Ser by Arg at residue 131. Genomic analysis of the family of case 2 by mismatched PCR showed that the missense mutation was consistent with their biochemical phenotypes. The replacement results in a conformational change of the residues near the Ser, probably because the side chain of Arg is much more bulky than that of Ser. The change may affect the arrangement of the cofactor binding site and result in the loss of enzyme activity. The experimental observations are consistent with computer graphics analyses.

97 High-Performance Liquid Chromatographic method for Simultaneous Screening of the Deficiencies of APRT and HGPRT

A rapid and covenient screening method using high-performance chromatography(HPLC) for the simultanous detection of deficiencies of adenine phosphoribosyltransf erase (APUT, EC 2.4.2.7) and hypoxanthine guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) is presented.The enzyme reactions were started by the addition of 0.1 ml of the red cell lysates (2∼5g Hb/dl) treated with charcoal-dextran into 0.5 ml of the reaction mixture A(2 mM PRPP, 6 mM MgCl2, 1 mM HX and 0.2 mM adenine in 50 raM Tris-HCl pH7.4). After incubation at 37°C, 30 min, 0.5 ml of reagent B (20 U/ml ALP, 4 mM MgCl2 in 1 M Tris) were added and incubated for 30 min. at 37°C for the conversion of AMP and IMP into adenosine and inosine. ALP reaction was stopped by adding 1.0 ml of 0.5 M HCIO4. The mixture centrifuged at 3,000 x g for 10 min. The supernatants were used as samples for HPLC analysis. Normal ranges of APRT and HGPRT activities in erythrocytes obtained 28 healthy subjects were 0.40 ± 0.06 and 1.97 μ 0.19 μ mol/min/ g Hb. respectively. The method could detect down to 1 % of normal APRT activity and 0.3 % of normal HGPRT activity.

Atypical fast-moving isozyme of alkaline phosphatase transiently found in sera of four children

An abnormal isozyme of alkaline phosphatase (ALP: orthophosphoric monoester phosphohydrolase, EC 3. 1. 3. 1) detected in the serum of four children with no malignancy is described.The molecular weights of the abnormal enzyme in all four patients were about 1.7×105 daltons, and the enzyme migrated to the front position of the liver ALP (α1∼α2 globulin region) irrespective of electrophoretic medium. Other features of the enzyme, i.e., Michaelis constants, molecular sizes, susceptibilities to amino acids and to the action of neuraminidase, and heat stability suggest that this enzyme is an anomalous bone ALP with respect to sugar chains.