Esterase and protease activity of purified guinea pig basophil granules.

Highly purified granules of circulating guinea pig basophilic leukocytes were extracted with 0.1% Triton X-100 to yield a mixture of esterases-proteases including caseinolytic activity. By selective inhibition both trypsin- and chymotrypsin-like serine hydrolases have been identified. Sigmoidal pH dependences for hydrolysis of p-tosyl-L-arginine-methyl ester and N-benzoyl-L-tryosine-ethyl ester were observed for both intact granules and Triton granule extracts. Preliminary studies indicate that the enzymes are not solubilized even after Triton X-100 treatment of the granules.     

The molecular stoichiometry of trypsin inhibition by human alpha-1-proteinase inhibitor

The stoichiometry of interaction of human alpha-1-proteinase inhibitor with porcine trypsin has been determined using a highly purified preparation of inhibitor. In contrast to the reports of others, one mole of alpha-1-proteinase inhibitor was found to inhibit two moles of trypsin. Disc gel electrophoresis indicates that the 2:1 complex is preferentially formed even when free alpha-1-proteinase inhibitor is still present.     

Serine protease inhibitors inhibit superoxide production by human basophils stimulated by anti-IgE

Anti-IgE-induced O₂^? production by human basophils was inhibited by potent inactivators of serine proteases. The inhibitory effect of the inhibitor and substrate for chymotrypsin-type protease was much greater than that of those substances for trypsin-type protease. These findings suggest that chymotrypsin-like serine proteases are involved in basophil O₂^? production.     

The sulfhydryl groups of the thiol-dependent cytolytic toxin from Bacillus alvei evidence for one essential sulfhydryl group

Alveolysin, an extracellular protein toxin (M_r ? 63,000) excreted by Bacillus alvei and purified to homogeneity was shown to contain four cysteine residues. All thiol groups of the hemolytically active toxin preparation were free as found by direct titration by 5,5′-dithiobis (2-nitrobenzoic acid) and confirmed by the absence of disulfide bond. Toxin alkylation with tosyl lysine chloromethyl ketone resulted in the complete loss of hemolytic activity and the disappearance of only one thiol group with no modification of histidine residues. These results support the conclusion that one essential thiol group is implicated in the membrane-disrupting activity of alveolysin.     

Interaction of serine protease inhibitors and substrates with human uterine estrogen receptor

The human uterine estrogen receptor has a site which regulates estrogen binding and which structurally resembles the substrate binding site of chymotrypsin. The hormone binding capacity and the affinity of the receptor is decreased in the presence of 4 mM serine protease inhibitors tosyl-lysine chloromethyl ketone and diisopropylfluorophosphate and the protease substrates tryptophan methyl ester and toluene sulphonyl-arginine methyl ester. The protease inhibitors tosylamide-phenylethyl-chloromethyl ketone and phenyl methyl sulphonyl fluoride caused an increase in the binding capacity whereas the affinity was decreased.     

Release and activation of phosphorylase phosphatase upon rupture of organelles from rat liver

Liver extracts (8000 × $\text{g}$ for 10 min) from fasted rats contain about 4 times more phosphorylase phosphatase activity when the liver was homogenized in a hypotonic medium or frozen before homogenization. This increase is caused by: (i) release of partially latent phosphatases ($\text{M}$_r=60 000 and 45 000 in sucrose gradient centrifugation) from ruptured organelles; (ii) rapid activation of phosphatase in the ruptured pellet by endogenous protease(s) which can be blocked by p-tosyl-L-lysine chloromethyl ketone. Only the $\text{M}$_r=60 000 enzyme, associated with the nuclei, can be activated proteolytically, with conversion to an $\text{M}$_r=45 000.