Takayuki Shiraishi

Protective protein in the bovine lysosomal beta-galactosidase complex.

Cathepsin A [EC 3.4.16.1], so called protective protein, occurs as an enzyme complex with lysosomal beta-galactosidase [3.2.1.23] and is involved in the stable enzymic expression of lysosomal sialidase [3.2.1.18]. In this study we investigated the enzymatic properties of cathepsin A in the bovine beta-galactosidase complex and how it is involved in the molecular multiplicities of the beta-galactosidase and sialidase complexes. Bovine protective protein homologous to the human protein had a molecular weight of 48 kDa on SDS-PAGE and cathepsin A activity optimum around pH 6.0. It hydrolyzed dipeptide substrates composed of hydrophobic amino acids much faster than any other type of substrate tested. This specificity was found to be conserved from human to a non-mammal, chicken. Immunoprecipitation using an anti beta-galactosidase antibody demonstrated that cathepsin A is a component of both the sialidase and beta-galactosidase complexes. The over 700 kDa sialidase complex depolymerized by a brief incubation at pH 7.5 and the sialidase was inactivated irreversibly via formation of an enzyme active smaller species of sialidase. The 669 kDa beta-galactosidase complex dissociated reversibly into a 120 kDa beta-galactosidase and a 170 kDa cathepsin A, but the 120 kDa beta-galactosidase, free from the cathepsin A, formed a 260 kDa aggregate under the same conditions. Inactivation of cathepsin A by heat treatment did not affect its complex forming activity. The 170 kDa protective protein dissociated into a 50 kDa one at pH 7.5, which no longer formed the complex. These findings indicate that the 170 kDa protective protein could be the minimum unit required for in vitro reconstitution of the complex, and that its complex forming activity is carried in a heat-stable domain. Both beta-galactosidase and cathepsin A activities were labile under the dissociated condition, indicating that it physiologically stabilizes not only beta-galactosidase but also itself by forming the complex.

Selective inhibition of lignoceroyl‐CoA synthetase by adenosine 5′‐alkylphosphates

Structural analogs of adenosine 5′‐acylphosphates, which are intermediates of the reaction catalysed by acyl‐CoA synthetases, were synthesized by condensing primary alcohols with AMP to examine the inhibitory effects on the lignoceroyl‐CoA and palmitoyl‐CoA synthetase activities. Hexadecyl, octadecyl, eicosyl, docosyl and tetracosyl esters of AMP were remarkably potent inhibitors of the lignoceroyl‐CoA formation. On the other hand, the eicosyl, docosyl or tetracosyl esters of AMP did not behave as significant inhibitors of the palmitoyl‐CoA formation at the concentration at which the two other shorter chain analogs were effective. Namely, these longer alkyl esters of AMP have selective inhibitory effects on the lignoceroyl‐CoA synthetase activity. The K i value of adenosine 5′‐tetracosylphosphate, the most potent inhibitor, was about one tenth lower than the K m value for the substrate lignoceric acid. Furthermore, the results support the notion that lignoceroyl‐CoA synthetase is distinct from palmitoyl‐CoA synthetase.

Effects of cyclodextrins on the hydrolysis of ganglioside GM1 by acid β-galactosidases

The hydrolysis of ganglioside GM1 by acid β-galactosidases was greatly enhanced by the inclusion of heptakis(2,6-di-O-methyl)-β-cyclodextrin or α-cyclodextrin in the assay mixture. The other cyclodextrins tested were not effective. The extent of stimulation by these cyclodextrins was relatively smaller than those by taurodeoxycholate and taurochenodeoxycholate. However, it is suggested that stimulation by bile salts may be partly a reflection of the detergent effects of bile salts on GM1 and partly a reflection of the interaction between bile salts and the enzyme itself. On the other hand, the stimulation by the cyclodextrins seems to correlate to the formation of an inclusion complex between GM1 and cyclodextrin without enzyme protein interaction.

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.

Studies on the Metabolism of Unsaturated Fatty Acids. III. Structural Determination of cis- and trans-Isomers of 3- or 2-Alkenoic Acids by Nuclear Magnetic Resonance Spectroscopy using a Chemical Shift Reagent

Nuclear magnetic resonance spectroscopy with a lanthanide chemical shift reagent was applied for the structural determination of cis-and trans-isomers of 3-alkenoic acids, prepared as substrates for some enzyme reactions related to fatty acid metabolism. The configuration of N-acetylcysteamine derivatives of cis-2-alkenoic acids was also determined by the same technique.