Yoshinori Kobayashi

Molecular cloning and expression of human chondroitin 6-sulfotransferase

Using cDNA of chick chondroitin 6-sulfotransferase (C6ST), human C6ST cDNA has been isolated. The amino acid sequence of human C6ST displayed 74% identity to chick C6ST. The major difference in amino acid sequence between chick C6ST and human C6ST was the presence of a unique hydrophilic domain in human C6ST. A 7.8-kb message of C6ST was expressed ubiquitously in various human adult tissues, indicating a rather diverse function of C6ST.

Purification and characterization of extracellular polyamine oxidase produced by Penicillium sp. No. PO-1

An extracellular polyamine oxidase produced by Penicillium sp. No. PO-1 was completely purified using the chromatofocusing method with a very high yield (93%) of the activity. The enzyme was composed of two identical subunits (Mr 64 000) and contained FAD. The optimal pH for activity was approx. 4.0. The enzyme oxidized spermidine and spermine. Km and Vmax values for spermidine were respectively 8.2 microM and 16.4 mumol H2O2/mg protein per min. Corresponding values for spermine were 5.3 microM and 13.3 mumol H2O2/mg protein per min. The enzyme attacked the secondary amino group of spermidine and spermine, and produced putrescine, 3-aminopropionaldehyde and H2O2. The enzyme activity was completely inhibited by phenylhydrazine. However, sulfhydryl reagents showed no effect on the activity. It is expected that the enzyme will be useful in determining the amount of polyamine in body fluids.

Oxidation of acetylpolyamines by extracellular polyamine oxidase produced by Penicillium sp. No. PO-1

The oxidation of acetylpolyamines by an extracellular polyamine oxidase of Penicillium sp. No. PO-1 was investigated. The optimal pH value for oxidation of acetylpolyamines was 6.0. The purified enzyme oxidized spermidine, spermine, N1-acetylspermidine, N8-acetylspermidine, N1,8-diacetylspermidine, N1-acetylspermine and N1,12-diacetylspermine. The relative velocities for oxidation of acetylpolyamines were lower than those of spermidine and spermine. The Km values for oxidation of acetylpolyamines were higher than those of spermidine and spermine. The enzyme split N1-acetylspermidine and N8-acetylspermidine at the same position of the linkage as in spermidine oxidation. N1-Acetylspermine was changed to N1-acetylspermidine. This oxidation mechanism was different from that of rat liver polyamine oxidase. N1-Acetylspermine inhibited the oxidation of spermine. Putrescine, N8-acetylspermidine and N1,12-diacetylspermine also inhibited the N1-acetylspermidine oxidation by the enzyme.

Purification and properties of NAD+-dependent maltose dehydrogenase produced by alkalophilic Corynebacterium sp. No. 93-1

Abstract NAD+-dependent maltose dehydrogenase was purified about 250-fold from the cell free extract of an alkalophilic Corynebacterium sp. No. 93-1. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultra-centrifugation. The molecular weight of the enzyme was determined to be 40 000 + 2000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme appeared to be a single peptide chain. The isoelectric point was pH 4.50. The optimal pH was 10.2. The enzyme was stable over the range of pH 6 to 10. NAD+-dependent maltose dehydrogenase showed very wide substrate specificity on monosaccharides, disaccharides and trisaccharides. Among these substrates, maltose was the most reactive. Also, the enzyme showed oxidative activity on maltotetraose and maltopentaose. The K m values at pH 10 were 2.1 mM for maltose and 0.15 mM for NAD+. It was conjectured that the primary product of this reaction was maltono-δ-lactone and it was hydrolyzed non-enzymatically to maltobionic acid. p- Chloromercuribenzoic acid , Hg2+ and Ag2+ completely inhibited the activity, and NADH also showed competitive inhibition on the activity.