Nagasumi Yago

Kinetics of suicide substrates steady-state treatments and computer-aided exact solutions

A steady-state differential equation that describes the kinetics of suicide substrate was derived for a scheme presented by Walsh et al. (Walsh, C., Cromartie, T., Marcotte, P. and Spencer, r. (1978) Methods Enzymol. 53, 437-488). Using its analytical solutions, the progress curves of substrate disappearance, product formation and enzyme inactivation were calculated for a hypothetical model system, and were compared with the exact solutions which were obtained by the numerical computation on a set of rate equations. The results obtained with the present analytical solutions were much more consistent with the exact solutions than those obtained using Waleys solution (Waley, S.G. (1980) Biochem. J. 185, 771-773). The most important factor for a system of suicide substrates was found to be the term (1 + r)mu as proposed by Waley, where r is the ratio of the rate constant of product formation to that of enzyme inactivation and mu is the ratio of initial concentration of enzyme to that of suicide substrate. In cases where this term has a value greater than unity, all the molecules of suicide substrate are used up leaving some enzyme molecule still active. To the contrary, in cases where the term has a value smaller than unity, all the enzyme molecules are inactivated with some molecules of suicide substrate being left unreacted. When the term is equal to unity, then all the enzyme molecules are inactivated and all the molecules of the suicide ar converted. Practical methods for estimating kinetic parameters are described.

Suppressive effects of tranilast on the expression of inducible cyclooxygenase (COX2) in interleukin-1β-stimulated fibroblasts

We investigated the effects of tranilast on inducible cyclooxygenase (COX2)-mediated prostaglandin E2 (PGE2) production and enzyme induction in interleukin-lbeta (IL-1beta)-stimulated cultured dermal fibroblasts. IL-1beta enhanced PGE2 production in cultured fibroblasts. Tranilast did not affect constitutive cyclooxygenase (COX1) or COX2 activity in non-stimulated or IL-lbeta-stimulated fibroblasts. However, the COX2 expression induced by IL-1beta was inhibited by tranilast. This result, that IL-1beta-induced COX2 expression was suppressed by tranilast, was confirmed by immunohistochemical analysis. Thus, it is possible for tranilast to regulate PGE2 production by inhibiting COX2 induction.

Polyphosphate anions increase the activity of bovine spleen cathepsin D.

Abstract Bovine spleen cathepsin D is activated by polyphosphate anions when bovine serum albumin is used as substrate at pH 4.6. In the presence of ATP at 10 mM, the catheptic activity at this pH is enhanced as high as 17 times over the control. Similar activating effects were observed, though to varying degrees, with sodium tripolyphosphate, nucleotides, nucleotide analogues, CoA, polyU and yeast RNA. The possible mechanism and biological significance of the activation were discussed with regard to the intralysosomal polyanionic substance.

Phospholipids activate cathepsin D

Total lipids as well as phospholipids extracted from the mitochondrial-lysosomal fraction of porcine adrenal cortex activated the lysosomal cathepsin D of this tissue 30- and 40-fold, respectively, with bovine serum albumin as the substrate. Phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol and cardiolipin were found to activate greatly the cathepsin D. The degree of activation ranged from 6-fold by phosphatidyl ethanolamine to 40-fold by cardiolipin at 1 mM, respectively. These results strongly point to the importance of phospholipids in intracellular protein degradation by lysosomal cathepsin D.

Activation of cathepsin D by polyanionic compounds

We examined the nature of the activation of cathepsin D by polyanionic compounds. Tripolyphosphate, a model compound for polyanions, decreased the Km value of porcine cathepsin D for bovine serum albumin without affecting Vmax. Half‐maximal activation was achieved at 0.2 mM free tripolyphosphate. Electrophoretic mobility of cathepsin D decreased as the tripolyphosphate concentration was increased, and the enzyme had no net charge at 50 mM triP. The concentration for the half‐maximal mobility change of cathepsin D (0.18 mM) was similar to that for half‐maximal activation. These results suggest that tripolyphosphate increased affinity of the enzyme for its substrate by cancelling positive charges on cathepsin D and thus decreasing the electrostatic repulsion.

Application of Three-Way Data Clustering to Analysis of Lymphocyte Subset Numbers in Japanese Hemophiliacs Infected with HIV-1

The 3-way data clustering was applied to the analysis of a number of CD4+ and CD8+ cells obtained from Japanese hemophiliacs infected with HIV-1 through non-heat treated clotting factor concentrates. A total of 131 hemophiliacs were classified into four clusters, termed Cluster 1, 2, 3 and 4. The members in Cluster 1 and 2 showed almost continuous decline in the number of CD4+ cells, while members in Cluster 3 and 4 showed unclear declining tendency compared to Cluster 1 and 2. The number of CD8+ cells was declining in Cluster 1, 2 and 3, while it was rising in Cluster 4. Nevertheless, the cumulative onset rates of AIDS in the four clusters cannot be divided into the four groups accordingly. The rate was the highest in Cluster 1 while no eminent differences were found among the other three clusters. Therefore, we could identify that there is a large variety in the time courses of CD4+ and CD8+ cell numbers during AIDS incubation period, even if the onset rates were almost identical. These findings may be helpful to find the clue in preventing the onset of AIDS arid selecting appropriate therapy for HIV-1 infected patients.