Yoshiko Usami

Decreased thymosin β4 in apoptosis induced by a variety of antitumor drugs

As many antitumor drugs can kill tumors through the induction of apoptosis, the effect of these drugs presumably would be enhanced if they were used in combination with other drugs that interact with apoptotic processes. To clarify the biological events involved in the induction of apoptosis, we examined changes in the proteins associated with induction of apoptosis by antitumor drugs. When Molt-4 cells were exposed to the antitumor drugs etoposide, meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane (ICRF-193), and neocarzinostatin, they exhibited apoptotic cell death as determined by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin V staining of phosphatidylserine on membranes and detection of hypodiploid cells. Following the induction of apoptosis, a low molecular weight protein that was identified to be thymosin beta4 by HPLC analysis was commonly decreased, and the morphology of actin filaments changed into clump formations. These results suggest that decreased thymosin beta4 is involved in the induction of apoptosis by antitumor drugs.

The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3

The cleavage site of human insulin‐like growth factor binding protein‐3 by urinary prostate specific antigen was examined. human insulin‐like growth factor binding protein‐3 was incubated with urinary prostate specific antigen at 37°C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N‐terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr‐159. The synthetic peptide including Tyr‐159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin‐like growth factor binding protein‐3 is specifically cleaved at Tyr‐159 by prostate specific antigen. human insulin‐like growth factor binding protein‐3 was previously reported to be cleaved at five sites including Arg‐97, Arg‐132, Tyr‐159, Phe‐173 and Arg‐179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin‐like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin‐like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin‐like growth factor binding protein‐3.

An Enzyme Immunoassay for Cuprozinc Superoxide Dismutase Using Monoclonal Antibodies. : Application for Pharmacokinetic Study.

We developed an enzyme immunoassay for determining human cuprozinc superoxide dismutase (h-SOD) using two kinds of monoclonal antibodies prepared by immunizing h-SOD to BALB/c mice. This method was sensitive and specific enough to determine exogenous h-SOD injected into rats. When intravenously injected into rats, much of the immunoreactive h-SOD accumulated in the kidney and was rapidly excreted in the urine. We observed both a modified and an unmodified form of exogenous h-SOD in rat urine.

Purification and partial characterization of an indomethacin hydrolyzing enzyme from pig liver.

AbstractPurpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of ρ-chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in α-naphthylacetate and ρ-nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.