Nobuaki Ogasawara

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80,000, isozyme M 72,000 and isozyme L 68,000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological an kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.

Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain

Abstract: Antisera were raised in rabbits against the α sub‐unit of a GTP‐binding protein, Go. Because the antisera cross‐reacted weakly with the α subunit of inhibitory GTP‐binding protein of adenylate cyclase (Gi), they were purified with a Goα‐coupled Sepharose column. Purified antibodies reacted only with Goα and did not cross‐react with the Giα subunit or βγ subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Goα was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab’ fragments labeled with β‐D‐galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Goa, and it did not cross‐react with Giα or βγ. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Goα were determined. Goα was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neu‐ropil was rich in Goα.

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns.

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.

Isozymes of rat AMP deaminase.

Three AMP deaminase isozymes (EC 3.5.4.6 AMP aminohydrolase) were purified from rat heart, kidney and muscle. These enzyme preparations contained only the required isozyme. Antisera to individual isozymes were prepared and immunological relationships were tested. There was no cross-reactivity as tested by precipitation experiments. The antisera precipitated only the corresponding isozyme and there was no effect on other isozymes. These isozymes were also different in Km values for AMP and in substrate specificity. From the present studies, combined with previous results, it seems clear that the heart, kidney and muscle enzymes are different basic types. It is proposed that the muscle enzyme be designated as AMP deaminase A; the enzyme in kidney and liver, AMP deaminase B; the enzyme in heart, AMP deaminase C. Brain extracts contained five isozymes; two parent isozymes (B and C) and presumably their three hybrids.

Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5′ end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5′ end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.

S-100 protein in clonal astroglioma cells is released by adrenocorticotropic hormone and corticotropin-like intermediate-lobe peptide.

Abstract: S‐100 protein in clonal GA‐1 and C6 rat glioma cell lines was released in serum‐free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S‐100 protein release by ACTH was dose‐dependent, showing a half‐maximal release at about 5 μM, and the S‐100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S‐100 protein release was apparently accompanied by a decrease in the membrane‐bound form of S‐100 protein in the cell. The S‐100 protein release was induced not by the ACTH1–24 fragment, which exhibits the known effects of ACTH, but by the ACTH18–39 fragment, which is designated as corticotropin‐like intermediate‐lobe peptide (CLIP). These results indicate that the C‐terminal half of ACTH is responsible for the S‐100 protein release. The enhancement of S‐100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S‐100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S‐100 protein release; however, catecholamines (epinephrine, norepi‐nephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release. The S‐100 protein release was also stimulated by 5 mM ATP, ADP, and GTP, probably by lowering the concentration of Ca2+ in the medium, because addition of Ca2+ inhibited the release by the nucleotides (but not by the cyclic nucleotides or ACTH), and 1 mM EGTA could induce the S‐100 protein release from GA‐1 glioma cells.

Pig mitochondrial DNA: polymorphism, restriction map orientation, and sequence data

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet.23:105]. mtDNA polymorphisms were observed in the cleavage patterns of five restriction enzymes, Bg1II, EcoRV, ScaI, StuI, and TaqI. The results support the previous hypothesis that pigs must be derived from two different maternal origins, European and Asian wild boars, and that a breed, Large White, arises from both European and Asian pigs. Two HindIII cleavage fragments were cloned into the HindIII site of M13mp10 and were partially sequenced by the dideoxynucleotide-chain termination method. Furthermore, DraI and StuI cleavage sites were newly determined on the restriction endonuclease map. On the basis of these results, the restriction endonuclease cleavage map of pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237 positions with those of cow, human, mouse, and rat mtDNA, the sequence difference, silent and replacement changes, and transitions and transversions among mammalian species were estimated. The relationships among them are discussed.

Enhancement of S-100β protein in blood of patients with down’s syndrome

The human gene encoding the β subunit of S-100 protein (S-100β) was mapped on chromosome 21. In order to confirm the expression of gene-dosage effect of S-100β in patients with Down’s syndrome (DS), concentrations of immunoreactive S-100α and S-100β proteins were determined in the blood plasma and lymphocytes fraction of the patients and control subjects. Cu/Zn-superoxide dismutase (SOD), a protein that is known to show the gene-dosage effect on the trisomy of chromosome 21, also was immunoassayed in the same blood samples as control proteins. In blood plasma, S-100β protein as well as Cu/Zn SOD was enhanced (P<0.001) in the patients (160±70 pg S-100β/ml and 87±83 ng SOD/ml, N=44) as compared with control individuals (76±25 pg/ml, and 18±11 ng/ml, respectively, N=28). However, concentrations of S-100α in blood plasma of DS patients were similar to those of normal subjects. Concentrations of S-100β in lymphocyte fractions of DS patients (24.7±10.9 ng/mg protein) were also higher (P<0.001) than those of control subjects (10.1±5.8 ng/mg protein). These results indicate that gene-dosage effect of S-100β levels are expressed in DS patients.

Induction of S100 protein in 3T3-L1 cells during differentiation to adipocytes and its liberating by lipolytic hormones

When confluent cultures of cloned mouse 3T3-L1 cells were differentiated to adipocytes by three days of treatment with a combination of 0.5 microM dexamethasone and 0.5 mM 1-methyl-3-isobutylxanthine, the S100 protein content in the cells increased markedly, as determined by a sensitive immunoassay system. The S100 protein induced in the cell was the alpha alpha form (S100ao), which is the predominant form of S100 protein in mouse adipose tissue. The S100ao concentration in preadipocytes was about 1-3 ng/mg protein, while the concentration in differentiated adipocytes was 60-200 ng/mg protein. The immunoblotting test of the crude extract of adipocytes confirmed that the immunoreactive substance in the cells was the alpha subunit of S100 protein. The treatment with 1-methyl-3-isobutylxanthine or dexamethasone alone neither elicited the S100 protein induction nor triacylglycerols accumulation in the cells. The accumulation of triacyglycerols in the cells was always preceded by the induction of S100ao protein under conditions where the differentiation to adipocytes was elicited. The induction of S100ao protein and accumulation of triacylglycerols in the cells treated with dexamethasone and 1-methyl-3-isobutylxanthine were inhibited by the addition of antimicrotubular drugs, colchicine and vinblastine, but not by cytochalasin B, an antimicrofilament drug. S100ao protein in 3T3-L1 adipocytes was released by incubation with a lipolytic hormone, adrenocorticotropic hormone or catecholamines, in a cyclic-AMP-dependent manner as observed with rat epididymal fat pads [Biochim. Biophys. Acta (1986) 889, 84-90]. These results also suggest that S100 protein may participate in the function of adipocytes.

Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain

Abstract: GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum, and hippocampus and then partially purified by gel filtration and characterized. The apparent molecular weights of all these receptors were determined to be 600,000–650,000 by gel filtration, the sedimentation coefficients being 11.0–11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to two classes of sites in fractions from all three regions, and [3H]flunitrazepam bound to one class of sites. A comparison of the ratios of Bmax for fiunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these three regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these three fractions, with similar potency. CL 218,872, however, inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 value and that in the hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64, and 0.58 for cerebellum, cortex, and hippocampus, respectively.