[Shigella endotoxin protein--its electrophoretic and serological properties]. / Belok éndotoksina shigell--élektroforeticheskie i serologicheskie svoistva.

The electrophoretic analysis of lipid A-associated protein (LAP), obtained from S. sonnei, in polyacrylamide gel in the presence of sodium dodecyl sulfate and urea has revealed the heterogeneity of the preparation; it has found to contain three main components with molecular weights of 43, 38 and 18 KD and some minor components with molecular weights of 49, 45 35, 30, 29, 27, 5, 21 and 14 KD. The electrophoretic mobility of the main protein components in the isolated preparation of LAP coincides with that of endotoxin components. The dissociation of proteins and lipopolysaccharide in the process of boiling the endotoxin in 2% sodium dodecyl sulfate is indicative of the noncovalent binding of these components. LAP contained in the endotoxin, in contrast to isolated LAP, is resistant to trypsin and proteinase K. The enzyme immunoassay (EIA) system with the use of LAP as a component of its solid phase has been developed, which makes it possible to carry out the quantitative determination of antibodies to this protein. The EIA system shows high sensitivity in the determination of anti-LAP IgG antibodies: in hyperimmune rabbit sera their titer is 1:250,000-1:800,000. As shown by the method of competitive EIA, the antigenic affinity of LAP of different origin corresponds to the degree of taxonomic propinquity of microorganisms: the maximal degree of cross reactions is observed between LAP obtained from S. sonnei, S. flexneri and Escherichia coli, while their affinity to Salmonella typhi is considerably less; remote microbial species (Bacterium bifidum and Sarcina marcescens) give practically no cross reactions.

[Various properties and kinetics of interaction of high and low molecular weight human kininogens with human plasma kallikrein]. / Nekotorye svoistva i kinetika vzaimodeistviia s vysoko- i nizkomolekuliarnymi kininogenami cheloveka kallikreina plazmy krovi cheloveka.

A relatively simple procedure for isolation and purification of human blood plasma kallikrein (HPK) by QAE-Sephadex A-50 SP-Sephadex C-50 and affinity chromatography on Sepharose 4B with immobilized soybean trypsin inhibitor with the activity yield of about 40% has been developed. The method allows for simultaneous isolation of low (LMW) and high molecular weight (HMW) kininogens from the same HPK sample. HPK preparations are homogeneous upon 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% SDS; its Mr is 90,000. After treatment with beta-mercaptoethanol, HPK dissociates into two fragments with Mr of 43,000 and 37,000. HPK preparations have high specific activities of esterase (31 microM/min), amidase (78 microM/min), and kininogenase (420 micrograms equiv. bradikinin/min). The high degree of protein purification was demonstrated by titration of active centers with 4-methylumbelliferylguanidine benzoate. The values of equilibrium dissociation constants for the HPK complex with aprotinin (Ki) equal to 1 X 10(-8) M (ethyl ester of N-alpha-benzoyl-L-arginine) and 1,5 X 10(-9) M (HMW) were determined. The kinetics of HPK-induced liberation of bradikinin from purified preparations of HMW and LMW was studied. The kinetic parameters (Km, kcat and kcat/Km) of this reaction suggest a high affinity of HPK for HMW, but not for LMW. LMW does not compete with HMW for the enzyme active center. It is assumed that LMW is not a physiological substrate for HPK.

[Characteristics of the kallikreins in healthy and malignant (adenocarcinoma) mucous membrane of the human large intestine]. / Kharakteristika kallikreinov zdorovoi i malignizirovannoi (adenokartsinoma) slizistoi obolochki tolstoi kishki cheloveka.

Total kininogenase activity was higher by 50% in supernatant of malignant mucosa homogenate as compared with normal tissue. But specific kininogenase and N-acetyl-arginine esterase activity were distinctly similar both in normal and tumoral tissues; N-ac-arg esterase activity tended to decrease in malignant mucosa. Partially purified preparations of kallikrein, isolated from adenocarcinoma and normal mucosa of human large intestine, were distinctly identical in their properties: both these enzymes were not inhibited by soybean trypsin inhibitor, they were highly sensitive to main pancreatic inhibitor from bovine tissues (Ki = 10(-10) M) and their molecular mass was about 40,000 daltons. The data obtained suggest that these enzymes may be concerned with tissue kallikreins. Highly purified preparation of kininogenase (85-90% of purity) first obtained from adenocarcinoma of human ascending colon, was identify with tissue kallikreins as it hydrolyzed low molecular kininogen, liberating the kinin--lysyl-bradykinin.

[Human low molecular weight kininogen: preparation, purification and immunochemical properties]. / Nizkomolekuliarnyi kininogen cheloveka: poluchenie ochishchennogo preparata, immunokhimicheskie issledovaniia.

A purified preparation of low molecular weight kininogen (LMWK) (Mr = 70 000+ +2 000) has been obtained from human blood plasma. The kinin content per 1 mg of the obtained preparation is 14--16 micrograms of bradykinin. A rabbit antiserum to LMWK which is free of antibodies against IgG and albumin present as admixtures in the kininogen preparation has been obtained. The titer of the antiserum as measured by dilution of antibodies, with normal human blood plasma as an antigen source, is 1 : 16. 1 ml of the antiserum binds 125 micrograms of purified LMWK in the equivalence zone. Immunoelectrophoretic studies of the antiserum to LMWK revealed three precipitation arcs with human plasma. It was assumed that these immunocomplexes are formed with a low and high molecular weight forms of kininogen and, possibly, with a high molecular weight kininogen complex with prekallikrein.

[Action of two thiol proteinases from the spleen which are active in neutral media on vasoactive peptides]. / Deistvie dvukh aktivnykh v neitral'noi srede tiolovykh proteinaz selezenki na vazoaktivnye peptidy.

The action of two earlier isolated highly purified spleen thiol proteinases on angiotensins I and II, bradykinin and kallidin was investigated. It was demonstrated that proteinase I which is apparently cathepsin L from bovine spleen brings about rapid inactivation of angiotensin II with a splitting of the Tyr-Ile bond and a formation of two tetrapeptides. Proteinases I also split angiotensin I. Proteinase I partially inactivates bradykinin and kallidin by splitting the Gly4-Phe5 bond. The activity of proteinase I toward angiotensin II is about 50 times higher than that toward bradykinin. The corresponding values of Km and V are 7.5 X 10(-5) M and 10.0 mumole/min/mg. The possible role of proteinase I in angiotensin II inactivation under physiological conditions is discussed. Proteinase II converts kallidin to bradykinin by splitting off the N-terminal lysine. Proteinase II causes partial inactivation of bradykinin by splitting of the Gly4-Phe5 and Phe5-Ser6 bonds of this peptide. Proteinase II possesses both aminopeptidase and endopeptidase activities and is therefore cathepsin H from spleen. Proteinase II does not split either angiotensin I or angiotensin II.

[Identification of kinins releasing by pig pancreatic kallikrein from rabbit low molecular weight kininogen]. / Identifikatsiia kininov, osvobozhdaemykh pankreaticheskim kallikreinom svin'i iz nizkomolekuliarnogo kininogena krolika.

Pig pancreatic kallikrein is found to release kinins from LMrK with the rate of 800 mkg. eq. of bradykinin per min, per 1 mg of the enzyme. This kininogenase reaction is not accompanied by a ddep fragmentation of the kininogen molecule, because only a high molecular weight fragment which Mr is practically equal to that of intact LMrK (55-60 000), and a peptide identified as bradykinin, are found in hydrolysate. In the same conditions human salivary kallikrein is found to form kallidin from LMrK.

[Purification and properties of high-molecular-weight rabbit kininogen]. / Ochistka i svoistva vysokomolekuliarnogo kininogena krolika.

A preparation of high-molecular weight kininogen (HMWK) was isolated from rabbit citrate blood plasma and purified using chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. From 1 mg HMWK, trypsin or kallikreine of human blood plasma release 10 mkg bradykinine. The HMWK preparation is homogeneous during electrophoresis in 7.5% polyacrylamide gel in tris-glycine buffer, pH 8.3; its electrophoretic mobility corresponds to that of alpha2-globulins. The molecular weight of HMWK estimated using the collumn with Sephadex G-200, is 130.000--150.000; the sedimentation constant S20w is 7.6. Rabbit HMWK is neither a dimer, nor a trimer of low molecular weight kininogen (LMWK), since it does not degrade into subunits after treatment by 2.5% solution of sodium dodecyl sulfate, containing 8 M urea. 0.05 M 2-mercaptoethanol and 8 M urea induce HMWK splitting into 2 fragments with respective molecular weights of 80.000 and 30.000, the kinine-containing group being localized in the low-molecular weight fragment. Estimation of rates of kinine formation by different kininogenasses from highly purified HMWK and LMWK preparations showed that those kininogens are functionally different substrates, since blood plasma kallikreines release kinines from HMWK at a greater rates, whereas tissue kallikreines, e. g. human saliva kallikreine release kinines from LMWK. The specificity of kallikreines as kininogenase, to trypsin, was determined. Tripsin removes bradykinine from both kininogens at the same rates, which are an order of magnitude less than those found for kallikreines.

[Amino acid makeup, structural characteristics and substrate specificity of rabbit plasma kininogen]. / Aminokislotnyi sostav, osobennosti stroeniia i substratnaia spetsifichnost' kininogena plazmy krolika

Highly purified kininogen preparation with the activity of 16-18 int. units per mg was isolated from rabbit blood serum. Its molecular weight was estimated to be 54 000 by gel filtration through Sephadex G-200. Leucine was identified as N-terminal amino acid by the dansylation method. Rabbit kininogen consists of 394 amino acid residues (except tryptophane). Amino acid composition of kininogen is characterized by a high content of dicarbonic amino acids, proline and by a low content of methionine. Kininogen molecule does not contain SH-groups. 13.1-13.5 SH-groups were found in kininogen after the reduction of S-S bonds with beta-mercaptoethanol in the presence of 8 M urea, thus indicating the presence of 6-7 S-S bonds in kininogen molecule. Kininogen group does not occupy C-terminal position in the molecule, because the treatment of the protein with carboxypeptidase B does not change the content of bradykinine in it. Purified kininogen preparation is a substrate for kallikrein from rabbit blood plasma, human saliva and trypsin. Unlike trypsin, kallikreines from human blood plasma and saliva release kinines from kininogen with reduced S-S bonds. Under spontaneous reoxidation of reduced S-S bonds up to 90%, substate properties of kininogen for tripsin recover only by 50%. Rabbit kininogen is similar to beef kininogen II in its molecular weight, amino acid composition and the number of S-S bonds.