Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.

Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.

Identification of reactive tyrosine residues in cysteine-reactive dehydrogenases. Differences between liver sorbitol, liver alcohol and Drosophila alcohol dehydrogenases.

Modification of tyrosine residues with tetranitromethane and reversible sulphite protection of cysteine residues were tested on three dehydrogenases of two families. In liver alcohol dehydrogenase no Tyr residue is appreciably labelled, while in the homologous sorbitol dehydrogenase Tyr-109 is specifically labelled; the difference corresponds to a segment correlating with subunit interactions and the different quaternary structures of the proteins. In Drosophila alcohol dehydrogenase, Tyr modification is multiple, and the results show the presence of two different states of Cys residues, reactive in the presence and absence of cupric ions, respectively. Super-activation with cyanide was also noticed after S-sulphocysteine protection. The results demonstrate the possibility of identification of specific Tyr residues in proteins with reversibly protected Cys residues.

Evidence that human ceruloplasmin molecule consists of homologous parts.

The products of spontaneous and induced proteolysis of human ceruloplasmin (Cp) were studied. Some physico-chemical properties of the six fragments with electrophoretically determined Mr 130,000 (F1), 110,000 (F2), 66,000 (F3), 48,000 (F4) 22,000 (F5) and 18,000 (F6) were compared. The amino acid compositions and N-terminal amino acid sequences coincide in F1-F5, but differ from those of F6. Limited tryptic proteolysis of Cp causes the accumulation of polypeptide fragment with Mr 22,000, the N-terminal primary structure of which is identical to that of F5 produced by spontaneous proteolysis. Electrophoretic fragments of Cp were extracted from polyacrylamide gel, treated with 125I and then studied by peptide mapping with subsequent radioautography. The comparison of the "finger prints" showed the identity of F1 to F2 and F3 and gross similarity between F4 and F1-F3. It also revealed similar peptides in F5 and F6 hydrolyzates and almost perfect matching of the F4 map to the map of F5 + F6 mixture. On the basis of the obtained data general principles of Cp molecular organization are discussed and intramolecular homology is suggested to be a feature of the protein.

[Comparison of anticholinesterase activity and the effectiveness of anticholinesterase substances]. / Sopostavlenie antikholinesteraznoi aktivnosti i effektivnosti antikholinesteraznykh sredstv

In vitro tests set up to compare the anticholinesterase, cholinomimetic, cholinopotentiating and cholinolytic activity of 6 reversible cholinesterase inhibitors furnished grounds for an inference that all these agents produce an anticholinesterase effect which determines the qualitative specificity of their action. This specificity has been demonstrated in experiments on cats and rabbits. Some of the drugs (oxazyl) exert a practically elective effect on the H-cholinoreactive muscle systems, whereas others (eserin) act in a similar manner on the M-cholinoreactive systems of the intestines. The action of proserine and galanthamine os of a less selective nature.