Search PubMed⌕ Search

Biomedical subjects

H Homma

Publications and source records attributed to H Homma.

At least 127 records · Page 7Linked to original sources
PubMed1987

Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets.

The binding profile of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) to washed rabbit platelets was investigated through the use of structural analogs of AGEPC, e.g. U66985, which specifically suppressed AGEPC biological activities on rabbit platelets. This interaction of AGEPC with platelets could be divided into three different components termed A, B, and C. Component A was considered as one of high affinity (Kd = 0.5 X 10(-9) M) and with a low capacity (about 400 sites/platelet). The binding of AGEPC to component A was reversible and was blocked by the inhibitory analogs of AGEPC. This was considered to be the AGEPC receptor site(s). Component B was irreversible in nature and was presumed to be associated with internalization of AGEPC. The latter process was sensitive to the structural inhibitors. Component C was not affected by the inhibitors and probably represented a nonspecific binding to the lipid layer of the membrane. The binding profile of 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine, a biologically inactive and noninhibitory analog of AGEPC, was observed to consist of a single component and was (also) unaffected by the inhibitors. Internalization of AGEPC into rabbit platelets was further examined by the bovine serum albumin extraction method, which was originally developed by Mohandas et al. (Mohandas, N., Wyatt, J., Mel, S. F., Rossi, M. E., and Shohet, S. B. (1982) J. Biol. Chem. 257, 6537-6543). AGEPC was instantly taken up by the cell and internalization into its membrane, where it remained and was not released into cytosol. The internalization of AGEPC was suppressed by pretreating the cells with AGEPC analogs. In platelets desensitized to AGEPC, no down-regulation of the receptor site(s) was observed. The internalization of AGEPC in the desensitized cells was clearly enhanced and this was obvious even in the presence of the AGEPC inhibitor(s). Even in the presence of the inhibitors, effective internalization of AGEPC was also evident in thrombin-treated cells. These results suggested that the internalization of AGEPC was irreversibly enhanced in the platelets which were activated by AGEPC itself as well as by thrombin.

Animals↗
PubMed1987

Some unique features of the metabolic conversion of platelet-activating factor (AGEPC) to alkyl acyl PC by washed rabbit platelets.

Recently several synthetic analogs of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor) were characterized as selective inhibitors of this agonist's effects on rabbit platelets (Tokumura, A., Homma, H., and Hanahan, D. J. (1985) J. Biol. Chem. 260, 12710-12714). In this current investigation, these studies have been extended to include a further inquiry into the biochemical nature of the metabolic inactivation of AGEPC in rabbit platelets, and the effect of these analogs on this process. Two of the latter components (U66985 and CV3988), which blocked AGEPC biological activity on rabbit platelets, also blocked the metabolism of this agonist. The metabolic conversion of AGEPC to alkyl acyl PC was inhibited nearly sevenfold by the most potent analog, U66985. Those analogs with low (U68043) or no biological inhibitory activity (lysoGEPC) had marginal effects on the metabolism of AGEPC. The effects of these compounds on the metabolism of AGEPC was not simply due to competitive inhibition. In platelets which had been pretreated with AGEPC in absence of extracellular Ca2+ (desensitized) and washed, the metabolic conversion of AGEPC to alkyl acyl PC was actually enhanced. This enhanced metabolic inactivation of AGEPC was also observed upon the treatment of the cells with thrombin, collagen, or ionophore A23187, indicating that the metabolism of AGEPC in platelets was enhanced not only by AGEPC itself but by other agonists as well. Nearly 85% of the fatty acyl residues was arachidonate in the alkyl acyl PC derived from AGEPC. This specific acylation with arachidonate was observed in the presence and absence of the inhibitor and in desensitized cells, indicating that selectivity for arachidonate is not dependent on the enhancement of the metabolism of AGEPC. The alkyl acyl PC found in the cells treated with thrombin, collagen, or A23187 was also predominantly alkyl arachidonoyl PC. Thus it has been shown that the inactivation of AGEPC by its conversion to alkyl acyl PC by rabbit platelets is enhanced by this agonist itself and that excess amounts of AGEPC could be further inactivated by the enhanced capacity of the metabolism process.

Animals↗
PubMed1987

Characteristics of phospholipid transacylase of Escherichia coli.

We have previously demonstrated the activity(ies) of phospholipid transacylase in Escherichia coli extract (Homma & Nojima (1982) J. Biochem. 91, 1093-1101), which catalyzed a new type of reaction of acyl transfer from diacylphospholipids to lysophospholipids. In this communication we report the specificities and characteristics of this enzyme activity. The activity catalyzed a reversible transfer of an acyl group between diacylphospholipids and lysophospholipids. The acyl group in the 1-position of the glycerol backbone was selectively transferred, and palmitic acid was the only fatty acid species transferred. Presumably, neutral lipids do not serve as substrates. The transacylase was firmly associated with the envelope fraction of E. coli. Neither potassium chloride nor urea was effective in solubilization of the activity and only about half of the activity was solubilized with Triton X-100. This observation was consistent with the equal distribution of the activity between the outer membrane and the inner membrane of E. coli. Functional aspects of this phospholipid transacylase are also discussed.

Acyltransferases↗
PubMed1987

A comparison of uridine diphosphate-glucuronosyl-transferase and other drug metabolizing enzyme activities between two mutant strains of Wistar rats with a genetic deficiency in bilirubin or androsterone glucuronidation.

A jaundiced rat strain was derived from a cross between Gunn and Wistar-Imamichi rats, and inbreeding was continued by forced heterozygosis with jaundice locus. These Gunn rats have black pigment on heads and a black stripe on their backs similar to Long-Evans rats. Wistar rats with low activity in androsterone (AD) glucuronidation were selected and inbred (LA Wistar rats). The levels of hepatic uridine diphosphate-glucuronosyltransferase (GT) and sulfotransferase (ST) activities as well as cytochrome P-450 contents were compared in these mutant strains. Gunn rats were devoid of bilirubin (BL) GT activity but had high AD GT activity. LA Wistar rats had very low AD GT activity but showed high BL GT activity. Native and Triton X-100-stimulated GT activities toward 2-aminophenol and 4-nitrophenol (NP) were much lower in Gunn rats than in LA Wistar rats. When N-nitrosodiethylamine was added to the incubation media, these GT activities were stimulated equally to high levels in both mutants. N-Nitrodiethylamine provided a similar stimulatory effect on NP GT activity. There were no significant differences in ST activities toward cortisol, AD and NP and cytochrome P-450 contents in the two mutant strains. These results indicate that Gunn and LA Wistar rats have a different deficiency in GT isoenzymes.

Androsterone↗
PubMed1986

A subclass of glutathione S-transferases as intracellular high-capacity and high-affinity steroid-binding proteins.

The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action.

Animals↗
PubMed1985

Structural analogs of alkylacetylglycerophosphocholine inhibitory behavior on platelet activation.

Several analogs of alkylacetylglycerophosphocholine (AGEPC; platelet-activating factor) were investigated as potential selective inhibitors of AGEPC-induced activation of washed rabbit platelets. Two particular compounds, CV-3988 (rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl-2-thiazolioethyl++ + phosphate) and U66985 (1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium-hexyl ester) emerged as particularly active and effective inhibitors. Aggregation and secretion profiles, as well as the degradation of inositol phospholipids and production of phosphatidic acid, were used as monitors of their inhibitory capabilities. U66985 was the most effective inhibitor, giving an IC50 value of 4.1 +/- 1.5 X 10(-8) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. Phospholipid turnover was blocked completely at this inhibitor concentration. On the other hand, while CV-3988 was an effective inhibitor, a higher concentration was required and a more restricted range of activity was noted with an IC50 value of 5.9 +/- 1.3 X 10(-7) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. While CV-3988 did indeed completely block the turnover of inositol phospholipids and phosphatidic acid formation, these effects were noted at a higher concentration than with U66985. On the basis of data obtained in desensitization experiments with AGEPC and U66985, it appears that each inhibitor occupies the same receptor site as the agonist, AGEPC. These results illustrate the usefulness of these AGEPC analogs in exploring the biochemical characteristics of the interaction of AGEPC with a cell.

Animals↗
PubMed1985

[Studies on preferential binding to glucocorticoid of rat liver anionic glutathione S-transferase].

A new isozyme of Glutathione-S-transferase (GST) with more acidic pI (6.7) than other forms of GST hitherto reported was isolated from rat liver cytosol by consecutive chromatographies on a DEAE cellulose column, lysyl-GSH affinity column and Sephadex G-100 column. This anionic form of GST represented approximately one third of total GST activity in rat liver cytosol. Amino acid composition, immunological reactivity, enzymatic properties, and secondary structure as measured by circular dichroism of this form were distinct from those of cationic isozymes (GST-AA, GST-B, GST-X), presently investigated. The stoichiometric ratio of high affinity site for bilirubin to GST molecule differs amongst isozymes. The anionic form of GST bound two bilirubin per molecule whereas cationic GSTs bound only one bilirubin per two subunits. The most distinguished property of anionic GST was its strong affinity for glucocorticoid. The dissociation constant of anionic GST-corticosterone complex was as low as 2.0 X 10(-8) M. Corticosterone inhibited the enzyme activity of anionic GST in a noncompetitive fashion with an apparent Ki value of 8.6 X 10(-5) M and 1.1 X 10(-6) M for 1-chloro-2.4-dinitrobenzene and GST respectively. The anionic GST-corticosterone complex bound to DNA coupled Sepharose at 25 degrees C and passed through the column at 4 degrees C. Conversely, the complex bound to a DEAE cellulose column at 4 degrees C but passed through at 25 degrees C. These properties of anionic GST are quite similar to those of glucocorticoid receptor of rat liver cytosol reported previously.

Amino Acids↗