Biomedical subjects
Publications and source records attributed to V V Mosolov.
The circular dichroism spectra of trypsin and chymotrypsin inhibitors from corn seeds were investigated. The spatial organization of the inhibitor molecules was shown to be different. The effects of guanidine chloride and temperature on these proteins suggest that the trypsin inhibitor from corn is much more stable towards the denaturating influences than the chymotrypsin inhibitor.
The protein proteinase inhibitor from kidney bean (isoinhibitor, pI 4.3) is a double-headed inhibitor with independent reactive sites for trypsin and chymotrypsin. The reactive site of the inhibitor for trypsin is Lys (22)- Ser (23) in the sequense ...-Lys-Ser-Ile-Pro-Ala-Glx-Cys-Arg-..., the reactive site for chymotrypsin is Leu (64)-Ser (65) in the sequence ...-Thr-Leu-Ser-Ile-Pro-Ala-Glx-Cys-Arg-... .
The trypsin inhibitor from Gleditsia triacanthos (L.) seeds was purified by affinity chromatography on a column with trypsin-Sepharose 4B. The isolated inhibitor is a single-chain protein with molecular weight of about 20 000. The inhibitor suppresses bovine trypsin at a molar rate of 1 : 1, but weakly inhibits chymotrypsin in a non-stoichiometric manner. Some properties of the isolated inhibitor closely resembled those of soybean trypsin inhibitor (Kunitz).
The chymotrypsin inhibitor was isolated from honey locust (Gleditsia triacanthos L.) by chromatography on trypsin- and chymotrypsin-Sepharose 4B and by gel filtration on Sephadex G-75. The inhibitor can inhibit chymotrypsin but has no effect on trypsin, subtilisin and proteinases from Asp. oryzae and Str. griseus. The inhibitor forms a complex with chymotrypsin at a molar ratio of 1:1, has a molecular weight of about 20,000 and a low content of half-cysteine.
Ovomucoid, a proteinase inhibitor from duck egg protein, was purified by affinity chromatography on trypsin-Sepharose. Isoelectric focusing revealed that the purified protein is represented by one major component with a pI of 3,8. The molecular weight of the inhibitor as determined by the sedimentation equilibrium method is 22 000 plus or minus 1000. One molecule of duck ovomucoid can bind two molecules of trypsin and one molecule of chymotrypsin. Modification of the protein by maleic anhydride showed that both centers responsible for trypsin binding contain lysine residues.
Using CD-spectroscopy and fluorescent probe technique, the formation of complexes of chymotrypsin with two highly efficient protein inhibitors of plant origin, e. g. inhibitors from pea seeds and potato tubers, was studied. The CD spectra recorded during the formation of the enzyme--potato tuber inhibitor complexes suggest that the inhibitor molecule. This is correlated with the data on fluorescence of ANS coupled to the inhibitor and to the inhibitor--chymotrypsin complex. In case of the pea seed inhibitor the changes in the CD spectra during the complex formation can result from the changes in the environment of the disulfide bonds in the protein--inhibitor molecule.
Treatment of a native protein inhibitor of proteinases by tetranitromethane results inmodification of 3 (out of 8) tyrosine residues in each of the two subunits within the inhibitor molecule. Nitration of surface tyrosines does not change the corformation of the protein and has no effect on its ability to inhibit chymotrypsin. At the same time the tetranitromethane-treated inhibitor possesses a decreased activity with respect to trypsin. In the presence of 0,5 M DS-Na practically all tyrosine residues of the protein are nitrated.
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The circular dichroism spectra of two protein proteinase inhibitors were studied. The CD spectrum of the kidney bean inhibitor is similar to those of other low molecular weight inhibitors from legume seeds. The potato inhibitor in its native state is characterized by a low content of alpha-helices, which is increased in the presence of sodium dodecyl sulfate.
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An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties.
The interaction between protein inhibitor of serine proteinases from potato (protein with pI 7.3) and enzymes was investigated. The main complex present in mixtures of the inhibitor with trypsin, chymotrypsin or subtilisin contained one dimeric inhibitor molecule and one molecule of either enzyme. At high ratio enzyme/inhibitor the complexes were formed which contained more than one bound enzyme molecule. The interaction with enzymes does not include the proteolytic degradation of inhibitor with concomitant lowering of its molecular weight. The inhibitor obtained after the dissociation of inhibitor--chymotrypsin complex, preserve its capacity to form stable complexes with the enzyme.
Two isoinhibitors (II and III-B) have been isolated from kidney bean (Phaseolus vulgaris L.) in a highly purified state. Both were active against trypsin and chymotrypsin to the same extent. Their amino acid composition is characterized by a high content of half-cystine, aspartic acid (or asparagine) and serine, by the absence of valine, methionine and tryptophan. Glycine and serine were N-terminal in II and III-B respectively. Both isoinhibitors have C-terminal leucine.
Crude hirudin preparation was purified by isoelectric focussing in pH gradient 3-5. The presence of at least two active isoforms with pI 3.8 and 3.9 was demonstrated. The component with pI value of 3.9 possessed the specific activity as high as 8200 NIH AT units/mg.
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