Different ratios in 20 S proteasomes and regulatory subunit complexes in two isoforms of the 26 S proteasome purified from rabbit skeletal muscle.

A ubiquitin/ATP-dependent proteinase complex (26 S proteasome) was highly purified from rabbit skeletal muscle. The purified 26 S proteasome easily dissociated into a 20 S proteasome and a regulatory subunit complex on non-denaturing PAGE. By using cleavable and non-cleavable cross-linkers, it was revealed that the 26 S proteasome exists in two isoforms: one (D complex) consists of the 20 S proteasome and the regulatory subunit complex in the ratio of one to two, while the other (C complex) exists in an equal molar ratio. Molecular masses of the former and the latter isoforms were estimated to be 1,700 kDa and 1,400 kDa, respectively, by gel filtration, and 2,400 kDa and 1,400 kDa, respectively, by Ferguson plot analysis. Furthermore, both isoforms efficiently hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and ubiquitin-conjugated [125I]lysozyme. These results suggest that the D and C complexes are active proteinase complexes, most probably corresponding to the dumbbell-like and mushroom-like (or space capsule-like) molecules, respectively.

Enhanced production of ethylene from methional by iron chelates and heme containing proteins in the system consisting of quinone compounds and NADPH-cytochrome P-450 reductase.

The addition of iron chelates or heme containing proteins to the systems consisting of NADPH-cytochrome P-450 reductase and quinone compounds, such as vitamin K3 (menadione), adriamycin, tetrahydropyranyladriamycin, daunomycin, aclacinomycin A, carbazilquinone, and mitomycin C, showed the enhanced production of ethylene from methional. In the vitamin K3 system, the effective iron chlates were Fe(II)-EDTA, Fe(II)-ADP, Fe(II)-bleomycin A2, and hemin, and the effective iron containing proteins were methemoglobin, myoglobin, ferredoxin, and partially purified cytochromes P-450, P-420, and b5, and the reversed effects were observed by horse radish peroxidase and sulfite reductase from yeast. In the system consisting of aclacinomycin A and methemoglobin, the ethylene production was potently inhibited by radical scavengers, such as Tiron, Tris, thiourea, and KI, and weakly inhibited by some other scavengers. In the system containing vitamin K3 and methemoglobin, the ethylene production was potently inhibited by catalase, but partially by superoxide dismutase, KCN, and NaN3. In this system, the absorption spectrum of methemoglobin was immediately changed to oxyform and quenched with time, and catalase protected the decrement of the spectrum. The addition of hydrogen peroxide or cumene hydroperoxide to methemoglobin also produced ethylene from methional.

Proton translocation coupled to nitrite reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli. Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells. This extrusion was sensitive to a proton conductor, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite. Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H+-ATPase. These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H+-ATPase. From the results obtained by the measurement of proton extrusion in nitrite reductase-deficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E. coli.

Enzymatic properties of sialidase from the ovary of the starfish, Asterina pectinifera.

A sialidase [EC 3.2.1 18] was isolated and highly purified from the ovary of the starfish, Asterina pectinifera, and its enzymatic properties were compared with those of human placental sialidase. The final preparation gave one broad protein band corresponding to sialidase activity on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 360000 by HPLC on Sigma Chrome GFC-1300 and Sephadex G-150 column chromatography, and 55000 by SDS-PAGE, suggesting the presence of a hexamer in the native protein. The optimum pH was between 3.0 and 4.0, and the enzyme liberated sialyl residues from the following compounds: alpha(2-3) and alpha(2-6) sialyllactose, colominic acid, fetuin, transferrin, gangliosides GM3, GD1a and GD1b. The enzyme was strongly inhibited by 4-aminophenyl and methyl thio-glycosides of sialic acid, but not by those glycosides of 5-amino sialic acid or sialic acid methyl ester. The enzyme was also highly inhibited by sulfated glucan and glycosaminoglycans. The substrate specificity and the effects of inhibitors on starfish sialidase were very similar to those of human placental sialidase.

A novel assay method for glycosphingolipid deacylase by enzyme-linked immunochemical detection of lysoglycosphingolipid.

Lysoglycosphingolipids consist of a sphingoid long-chain base and monosaccharide or complex sugar, and they lack the fatty acyl group present in native glycosphingolipids. Less than 1 pmol of lyso-Forssman glycolipid and lysoganglioside GM1 were detected on a thin-layer chromatogram by an enzyme-linked immunochemical coloration method with anti-Forssman glycolipid antibody (FOM-1) and cholera toxin B subunit, respectively. Each spot between 1 and 100 pmol lyso-Forssman glycolipid was immunostained as densely as that of the same amount of native Forssman glycolipid. The density of the lyso-Forssman glycolipid spots increased proportionally with increment in the amount of lysoglycolipid. The density of spots of 0.2-100 pmol lysoganglioside GM1 was also proportional to the amount of each lyso-GM1 spot. These results indicated that less than 1 to 100 pmol of deacylated glycosphingolipid was quantifiable by the immunochemical coloration method with sugar chain-specific antibodies. Glycosphingolipid deacylase, which cleaved an amide bond between the sphingoid long-chain base and fatty acyl chain in ceramide of glycosphingolipid, was assayed by detecting the lyso-Forssman glycolipid produced. Lipophilic compounds, recovered from an aliquot of the reaction mixture of Forssman glycolipid and crude enzyme at appropriate times, were analyzed by thin-layer chromatography. It was found that lyso-Forssman glycolipid was produced in the first 1-2 h by the enzyme and production increased with incubation time. This coloration method is more sensitive and specific than the visualization method with a nonspecific reagent such as orcinol-sulfuric acid reagent.

Participation of 650-kDa protease (20 S proteasome) in starfish oocyte maturation.

A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.

The 26S proteasome assembly is regulated by a maturation-inducing hormone in starfish oocytes.

Changes in proteasome activities were observed during starfish oocyte maturation induced by a maturation-inducing hormone, 1-methyladenine. Succinyl-Leu-Leu-Val-Tyr-MCA-hydrolyzing proteasome activity in immature oocytes showed a main peak of a 1500-kDa fraction and a shoulder centered at a 650-kDa fraction on Superose 6 gel-filtration chromatography in the presence of ATP and glycerole. The 1500-kDa activity transiently decreased and then increased at about a half the time required for germinal vesicle breakdown (GVBD). In contrast, the 650-kDa activity showed only a slight change during the maturation process. The activity of the 1500-kDa complex, unlike that of the 650-kDa complex, was immunoprecipitated with an antibody raised against regulatory subunits of mammalian 26S proteasomes, whereas both 1500- and 650-kDa activities were immunoprecipitated with anti-20S proteasome antibody. In addition, the 1500-kDa complex showed an ATP/ubiquitin-dependent proteolytic activity. These results indicate that the 1500- and 650-kDa complexes correspond to the mammalian 26S and 20S proteasomes, respectively. Immunoblot analysis revealed that the change in the 26S proteasomal activity is due to the change in the amount of the 20S proteasome subcomplex. Taken together, the proteasome undergoes changes in molecular assembly and activities during hormone-induced oocyte maturation.

Inhibition of starfish oocyte maturation by leupeptin analogs, potent trypsin inhibitors.

Inhibition of subsite-substituted leupeptin analogs, potent trypsin inhibitors, on 1-methyladenine-induced germinal vesicle breakdown was investigated in a starfish, Asterina pectinifera. Of benzyloxycarbonyl(Z)-Leu-P2-argininals, the analog with Ser at P2 residue was the strongest inhibitor, and those with Pro, Leu greater than Thr greater than Gly were followed in this order. In Z-P3-Ser-argininals, ranking of the inhibitory ability was as follows: Phe greater than Leu much greater than Pro greater than Ala at P3 residue. Among 11 analogs synthesized, Z-Phe-Ser-argininal showed the strongest inhibition. The inhibitory potency of the analog was 100-fold stronger than that of leupeptin (acetyl-Leu-Leu-argininal). Thus, trypsin-like enzyme possessing a narrow subsite specificity participates in oocyte maturation in the starfish.

Protease triggers dephosphorylation of cdc2 kinase during starfish oocyte maturation.

We previously presented evidence that a Z-Phe-Ser-argininal-susceptible protease which is involved in oocyte maturation of the starfish, Asterina pectinifera is the proteasome (Takagi Sawada et al, Dev. Biol. 150, 414-418 (1992)). In the present study, we investigated the timing of the function of and the role of the protease in oocyte maturation using Z-Phe-Ser-argininal. By adding the inhibitor in maturing oocytes at various times after 1-methyladenine treatment, the inhibitory ability was markedly reduced in half the time required for germinal vesicle breakdown. Furthermore, the inhibitor potently blocked the activation of histone H1 kinase and the dephosphorylation of cdc2 kinase during oocyte maturation. These results indicate that the Z-Phe-Ser-argininal-susceptible protease, probably the proteasome, plays a key role in the step of the signal transduction pathway that triggers the dephosphorylation of cdc2 kinase in response to the maturation-inducing hormone.