Shunji Sawaki

Detection of dipeptidase and tripeptidase activities on polyacrylamide gel and cellulose acetate gel by the reduction of tetrazolium salts

Abstract This paper describes the detection of dipeptidase and tripeptidase activities after polyacrylamide gel and cellulose acetate gel electrophoresis. This method, based on the reduction of tetrazolium salt, is easy and does not diffuse the way previously described methods have done. The peptidase activity of human liver and kidney on cellulose acetate gel appeared as two or three bands using l -Leu-Gly-Gly or l -Leu- l -Leu as substrate. Using l -Leu- l -Leu as substrate on polyacrylamide gel (7.5% gel, pH 9.4), peptidase activity appeared as one band in the liver and two bands in the kidney.

A simple one-step purification of human α1-proteinase inhibitor by immunoadsorbent column chromatography☆

A one-step purification of human alpha 1-proteinase inhibitor was described using the rabbit anti-alpha 1-proteinase inhibitor antibody coupled to CNBr-activated Sepharose 4B. The elution of alpha 1-proteinase inhibitor from the immunoadsorbent column using 0.1 M Na2CO3/0.5 M NaCl solution gave an 85% yield. The properties of eluted alpha 1-proteinase inhibitor were identical with that of alpha 1-proteinase inhibitor that was purified by the conventional method. In addition, the specific activity of purified alpha 1-proteinase inhibitor was more than 93% of that of the theoretical value.

Purification and properties of human liver peptidase

The peptidase from human liver was purified using L-Leu-L-Leu as a substrate, in adapted purification techniques including treatment with n-butanol, acetone precipitation, ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration and CM-cellulose chromatography. The purified enzyme exhibited homogeneity in disc electrophoresis. The molecular weight of the enzyme was estimated to be 130 000 by Sephadex G-200 gel filtration. The isoelectric point of the enzyme was found to be pH 5.6. The enzyme was activated by Mn2+ and inhibited by o-phenanthroline. L-Leu-L-Leu and L-Phe were hydrolyzed effectively by the peptidase. By electrophoresis on Cellogel, the electrophoretic mobility of purified enzyme was same as that of the peptidase in serum of patients with hepatic disease.

Purification and properties of human kidney dipeptidases.

Abstract Two dipeptidases from human kidney were purified with l -Leu- l -Leu as a substrate, and polyacrylamide gel (pH 9.4) electrophoresis showed that the peptidase obtained were homogeneous. On the basis of their electrophoretic mobilities the peptidases were classified as peptidases F (fast) and S (slow). The isoelectric point of both enzymes was estimated to be p I 4.7. The molecular weights of peptidase F and S by Sephadex G-200 gel filtration were estimated to be 135 000 and 200 000, respectively. From the result of SDS polyacrylamide gel electrophoresis, it was found that peptidase F consisted of two subunits with equal molecular weights of 66 000 and that peptidase S consisted of two subunits with molecular weights of 94 000 and 115 000. Both enzymes were stained with periodic acid-Schiff reagent in polyacrylamide gels, indicating that the enzymes were glycoproteins. Thermal stabilities of the two peptidases were very different. On heat treatment, peptidase F was stable up to 60°C but peptidase S became unstable above 40°C. Substrate specificities of the two peptidases were similar, and the enzymes hydrolyzed dipeptides such as l -Ala- l -Ala, l -Met- l -Met, Gly- l -Leu, l -Leu-Gly, l -Phe- l -Tyr, Gly-Gly and l -Leu- l -Leu to a significant extent.

Enzyme immunoassay of human cytosolic aspartate aminotransferase

A sensitive and specific sandwich enzyme immunoassay (EIA) for human cytosolic aspartate aminotransferase (c-AST) has been developed. Serum was incubated with anti-c-AST antibody-coated polystyrene beads, and further incubated with anti-c-AST antibody-peroxidase conjugate. The peroxidase activity bound to the polystyrene bead was proportional to the amount of c-AST. The method allows measurement of serum c-AST ranging from 50-2,000 micrograms/l. No cross-reactivity with m-AST or other serum components was observed. Recovery, within-day precision, and day-to-day precision were good. The levels of c-AST obtained by the proposed EIA were compared with those based on enzyme activity. The results suggest that there is a considerable excess of immunologically active but catalytically inactive c-AST in normal and patients sera, and that variable specific activities of c-AST are may be found in sera from different individuals.

Correlation between dipeptidase activity and concentration of human liver dipeptidase in serum

Abstract We describe the relationship between serum enzyme activity and concentration of human liver dipeptidase (HLD) by radioimmunoassay. Using the technique of differential centrifugation, it was found that dipeptidase was predominantly localized in the cytoplasmic fraction of human liver cells. Serum dipeptidase activity was 10.3 ± 2.3 I.U./l (mean ± S.D.) in normal subjects and increased remarkably in patients with diseases of the liver such as acute hepatitis (63.2 ± 27.8 I.U./l), cancer of the liver (123 ± 152 I.U./l) and pancreatic disease (68.0 ± 18.1 I.U./l). On double immunodiffusion and immunoelectrophoresis, using sera of patients with diseases of the liver and antibody to HLD, a single diffusion line was seen. Using double antibody radioimmunoassay, serum HLD concentration was 13.3 ± 7.3 ng/ml in normal subjects, and elevated in sera of patients with diseases of the liver, such as acute hepatitis (191 ± 146 ng/ml), cancer of the liver (171 ± 106 ng/ml) and chronic hepatitis (84.7 ± 86.1 ng/ml). There was a significant correlation between serum HLD concentration and serum dipeptidase activity ( r = 0.930); however, sera from four cases with pancreatic diseases and two cases with chronic hepatitis did not show this correlation.