Hirozi K. Kihara

Microassay of protein with nitrocellulose membrane filters

Abstract In the presence of 0.1 M NaCl and MgCl 2 , protein was found to bind quantitatively with nitrocellulose membrane filters. Staining the bound protein with amido black solution made possible the determination of protein from 5 to 50 μg, at a concentration as low as 0.01 μg/ml.

Regional differences of proteins in isolated cells of early embryos of Xenopus laevis

Regional differences of proteins were studied by two-dimensional gel electrophoresis in early embryos of Xenopus laevis. Pairs of blastomeres on the dorso-ventral axis were isolated from 16- and 32-cell embryos. Some dorso-ventral differences have been detected at 32-cell embryos. The proteins which were clearly detectable in the vegetal cells of the ventral marginal zone were only faintly detectable or undetectable in those of the dorsal marginal zone, and a regionally specific spot was detected in dorsal blastomeres.

Effects of Endopeptidase Inhibitors on Gastrulation of Xenopus Eggs

Protease inhibitors were injected into Xenopus embryos to determine the involvement of proteases in development. Injection of inhibitors of endopeptidase into the dorsal region of Xenopus embryos resulted in abnormal gastrulation with incomplete invagination and development of the embryos into defective tadpoles. In contrast, injection of protease inhibitors into the ventral region resulted in normal development. The frequency of abnormal gastrulation was significantly less on injection of an endopeptidase inhibitor in which the functional aldehyde group has been reduced with NaBH4. These results suggest that during early development of Xenopus embryos, the protease activity in the dorsal region is necessary for gastrulation.

Effects on properties of a thiol protease from Xenopus embryos of changes in substrate and assay conditions.

A protease was purified from Xenopus embryos. Proteolytic activity of the protease against BSA had an optimum pH of 3.8 in acetate buffer and was not detectable at neutral pH. However, when embryonic proteins were used as substrates and digested in phosphate buffer, proteolysis of embryonic proteins was enhanced and was detectable from pH 5.0 to pH 7.0. Digestion of three proteins were mainly detected in digestion of total embryonic proteins. The proteins digested had the same mobilities (on SDS polyacrylamide gel) as yolk proteins. The protease was present in the cytoplasm and around yolk granules. We propose that this protease mainly cleaves a certain yolk proteins in the cytoplasm of Xenopus embryos.

Characterization of proteolytic activities in embryos of Xenopus laevis

1. Proteolytic activities in early embryos of Xenopus laevis exhibited maximum levels at pH 3.2, 5.6 and 7.2 when 3H-BSA was used as substrate, and the maximum proteolytic activity at pH 3.2 was several thousand-fold higher during the tail bud stage than in the unfertilized egg. 2. The proteolytic activity at pH 3.2 was separated into two fractions by gel chromatography. One fraction corresponded to a mol. wt of about 40,000 and its activity was inhibited by thiol protease inhibitors. The other appeared to be a protease of much higher mol. wt. 3. The maximum activities at pH 5.6 and 7.2 appear to correspond to proteins of mol. wt greater than 1,000,000.