Specific cleavage of hybrid proteins by proteinase encoded by the KEX2 gene.

A method for isolation of the KEX2-gene-encoded membrane-bound proteinase from alpha-cells of Saccharomyces cerevisiae yeast has been modified. The isolated enzyme hydrolyzes peptides and proteins with basic amino acid pairs which are cleaved at the C-ends of their peptide bonds. Because KEX2 proteinase is located within the Golgi compartment, it may be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min. By extracting the fraction that contains the active enzyme by a detergent solution, a protein has been obtained with specific activity 30 times higher than that of the membrane extract prepared according to the standard technique. This protocol decreases the number of steps required to isolate the enzyme. The effects of pH and inhibitors on KEX2 proteinase-catalyzed hydrolysis of Ac-Leu-Lys-Arg-pNA were studied. KEX2 proteinase can participate in peptide hormone processing because it cleaves human proinsulin at the peptide bond between Arg32 and Glu33. The KEX2 proteinase can specifically cleave large recombinant proteins, for example, a protein consisting of a gamma-interferon fragment linked to HIV1-proteinase via a Lys-Arg-containing peptide.

Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Purification and properties.

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 +/- 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 +/- 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9-8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an active-site serine in this protease. alpha-N-Tosyl-L-lysine chloromethane and alpha-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly suppressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.

[Enteropeptidase and its use for cleavage of chimeric proteins]. / Enteropeptidaza i ee primenenie dlia rasshepleniia khimernykh belkov.

Cleavage of different chimeric proteins after specific linker (Asp)4Lys by the highly purified enteropeptidase was investigated, proteins being were accumulated in inclusion bodies or secreted from the cell. Kinetic constants for enzymatic hydrolysis were obtained, indicating that the substrate binding depended mainly on the affinity to the linker peptide (Asp)4Lys. Conditions for the efficient cleavage of recombinant proteins with enteropeptidase are formulated.

[ATP-dependent proteinase La from Escherichia coli]. / ATP-zavisimaia proteinaza La iz Escherichia coli.

Homogeneous preparations of the ATP-dependent La proteinase from E. coli and two its mutant forms, containing an alanine residue instead of Ser679 or Ser368, were isolated. Ser679 was shown to be catalytically active rather than Ser368 as suggested in the literature. To choose between the alternative structures of the gene lon La proteinase fragments within the controversial regions were analysed and the gene structure established at the Laboratory of Proteolytic Enzymes (Institute of Bioorganic Chemistry) was confirmed. Inactivity of La proteinase in some in vitro systems suggests its functioning in vivo to be not autonomous, requiring additional factors.

Identification, sequence analysis, and characterization of cDNA clones encoding two granzyme-like serine proteinases from rat duodenum.

Clones of cDNA encoding two serine proteinases were isolated from a cDNA library prepared from rat duodenum mRNA. The deduced amino acid sequences consisted of 248 residues and possessed a high level of homology to one another and to the sequences of granzymes, cathepsin G, and mast cell proteases I and II. Analysis of the enzymes' primary structures allowed the identification of the catalytic amino acid triad and the prediction of the substrate specificity. Northern blotting experiments showed that while one of these proteinases is expressed only in duodenum, the other enzyme is present in duodenum, lung, and spleen. It is supposed that these proteinases may play an important role in the function of an organism's defence systems.

[Amidation of proteins and peptides with carboxypeptidase Y]. / Amidirovanie belkov i peptidov s pomoshch'iu karboksipeptidazy Y.

The native and modified carboxypeptidase Y-catalyzed reaction of acyl transfer of acylamino acid and peptide residues from the corresponding esters to ammonia was studied. Use of the modified carboxypeptidase Y increased the yield of the resulting product. Calcitonin-Leu was transformed into human calcitonin.

[Cloning and expression of the 3C-proteinase gene from the poliomyelitis virus in E. coli cells]. / Konirovanie i ékspressiia gena 3C-proteinazy virusa poliomielita v kletkakh E. coli.

Expression of the 3C protease gene of poliovirus type 1 (Mahoney) in E. coli cells using various vectors was studied. The 3C gene was shown to be expressed effectively upon its cloning in HindII/HindII (bases 5240 to 6770) and in HindII/HindIII (bases 5240 to 6056) fragments of poliovirus cDNA in pTTQ8 vector containing tac-promoter and lacI-repressor gene. Products of processing at the N-terminal 3C protease Gln-Gly site and polypeptides formed upon translation from an alternative methionine, which was coded by bases 5516-5518 of poliovirus cDNA, were found among virus-specific proteins. Processing at the C-terminal 3C protease Gln-Gly site was not observed.

[Isolation and purification of 3C-proteinase from the poliomyelitis virus. Identification of an active form of the enzyme]. / Vydelenie i ochistka 3C-proteinazy virusa poliomielita. Identifikatsiia aktivnoi formy fermenta.

All the forms of 3C protease previously found were isolated and purified. A 3D polymerase's fragment covalently bound with 3C protein does not affect the specific proteolytic activity of the enzyme, whereas the elimination of 26 N-terminal amino acid residues of 3C protease leads to its inactivation.

Site-directed mutagenesis of La protease. A catalytically active serine residue.

Comparative sequence analysis of Escherichia coli ATP-dependent La protease led to the suggestion that Ser679 is the catalytically active enzyme residue. Site-directed mutagenesis Ser679----Ala, investigation of the cells containing the mutant plasmid, and study of the partially purified mutant protein produced results in favour of this suggestion.

Recombinant poliovirus 3C protease. The enzyme application to protein specific fragmentation.

Active 3C protease of poliovirus 1(M) was obtained when cloning and expressing fragment HindII-HindIII (bases from 5240 to 6056) of cDNA in vector pTTQ8 in E.coli cells. As shown, fragment 3D of polymerase covalently bound to 3C does not deprive the enzyme of its specific proteolytic activity. The absence of 26 N-terminal amino acids in 3C entails its inactivation. The recombinant 3C protease cleaved peptide bond Gln-Gly not only in virus polyprotein, but also in molecules of beta-galactosidase and bovine catalase.

Inorganic supports coated with N-substituted polyacrylamides: application to biospecific chromatography of proteins.

Wide porous glass (WPG) chemically coated with a poly-N-(2-hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the gamma-aminopropyl derivative followed by chemical adsorption of poly(p-nitrophenyl acrylate). Ester groups of the polyacrylate-coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly-N-(2-hydroxyethyl)acrylamide-coated support with non-specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one-step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.

[Cloning, structure and expression of the full-size lon gene in Escherichia coli coding for ATP-dependent La-proteinase]. / Klonirovanie, struktura i ékspressiia polnorazmernogo gena lon Escherichia coli, kodiruiushchego ATP-zavisimuiu La-proteinazu.

Assembly of the full Escherichia coli K-12 lon gene from the EcoRI--SphI fragment of the bacterial DNA ("modified" gene) cloned and sequenced earlier and the PstI fragment of the same DNA containing 3'-terminal region of the lon gene has been performed. Both "modified" and full genes showed all phenotype properties of lon gene. The complete nucleotide sequence of the gene (2770 bp) coding for the 784 amino acid sequence of protease La was determined. Location of catalytically active serine, histidine and aspartic acid residues was suggested, and ATP-binding site found. The lon gene and protease La structures we found are compared with those described independently and differences observed are discussed.

Regulation of intracellular proteolysis in Escherichia coli cells by antisense mRNAs of the lon gene.

To explore the possibility of regulating proteolytic processes in Escherichia coli cells, a series of plasmids directing the synthesis of antisense mRNAs complementary to different regions of the lon gene was constructed. The effect of these antisense mRNAs on the rate of degradation of abnormal proteins (14C-labelled polypeptides containing puromycin or canavanine) in the bacterial cells was studied. The greatest inhibitory effect occurred in the case of a mRNA complementary to the regulatory region at the 5' end of the lon gene. The reasons for the differences in the efficiency of lon gene antisense mRNAs in inhibiting proteolysis are considered.

[Complement-inhibiting acidic factors from the venom of Central Asian cobra Naja naja oxiana]. / Kislye faktory iada sredneaziatskoi kobry Naja naja oxiana, ingibiruiushchie komplement.

Two anticomplementic factors isolated from the venom of the Central Asian cobra Naja naja oxiana by chromatography on DEAE-Sepharose CL-6B and subsequent gel filtration on Sephacryl S-200 were studied. Of these, five factors (CFA-Ia, CFA-Ib, CFA-Ic, CFA-IIa and CFA-IIb), CFA-Ib had been characterized earlier, while CFA-Ia was assigned to a previously identified H-CoF factor. It was shown that CFA-Ic has a molecular mass of 3900 Da; its content in the venom amounts to 2.6 mg/g of dry venom. This factor inhibits the classical pathway of C3 convertase formation abrogating the C2 component activation by subcomponent C1s [Ki = (2.5 +/- 0.8).10(-7) M]. CFA-IIa and CFA-IIb are present in the venom in very low amounts (2 mg/g) and have Mr of 5700 and 3200 Da, respectively. The complement-inhibiting action was studied for a more active CFA-IIa. Factor CFA-IIa was shown to inactivate the native component of C2 with a rate constant, k, of (2.7 +/- 0.2).10(3) s-1M-1 (37 degrees C, pH 7.4). CFA-IIa had no effect on C2 and C2a within their complexes with C4b.

[Major complement inhibiting factors from the venom of the Central Asian cobra Naja naja oxiana]. / Osnovnye faktory iada sredneaziatskoi kobry Naja naja oxiana, ingibiruiushchie komplement.

The properties of two anticomplementic factors isolated by CM-Sepharose chromatography from the basic non-adsorbed on DEAE-Sepharose fraction of the Central Asian cobra Naja naja oxiana venom, were studied. Of these three factors (CFB-I, CFB-II and CFB-III) the latter had been characterized earlier. CFB-I was shown to be a protein with an N-terminal Asp and a molecular mass of about 39 kDa (data from gel chromatography); its content in the venom is 3.6 mg/g of dry venom. The protein inhibits mainly the classical pathway of the complement activation, being bound to component C4 (Ki = 9 nM). CFB-I seems to be analogous to the CI inhibitor from the venom of the Naja haje cobra. An analysis of the N-terminal sequence of CFB-II showed it to be identical to the earlier characterized cytotoxin I. CFB-I inhibits the formation of C3 convertase with Ki = 2.2-2.8 microM by way of binding to C4b and thus interfering with the component C2 sorption.

[Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent]. / Stupenchataia dissotsiatsiia subkomponentov C1, pervogo komponenta komplementa cheloveka, pri aktivatsii na affinnom sorbent.

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.

[Regulation of proteolytic processes using antisense RNA genes htpR and lon of Escherichia coli in hetero- and homologous genetic systems]. / Reguliatsiia proteoliticheskikh protsessov s pomoshch'iu antismyslovykh RNK genov htpR i lon Escherichia coli v getero- i gomologichnykh genetichestkikh sistemakh.

Possibility of correction of proteolytic processes in cells of Escherichia coli and Pseudomonas aeruginosa has been studied. For this purpose recombinant plasmids directing the synthesis of antisense RNAs were constructed. In Ps. aeruginosa the synthesis of htpR antisense RNA resulted in 2.5-fold reduction of the intensity of degradation of 3H-puromycin polypeptides under heat shock conditions. An antisense RNA complementary to the 5'- end of E. coli lon gene decreased the same index to the level observed in lon- mutants. Genes homologous to htpR and lon genes of E. coli were found in Pseudomonas: bacteria in hybridisation experiments. This finding suggests that the genetic system of heat shock in these microorganisms is organized in a similar manner.

Catalytic activity and association of pancreatic lipase.

The authors summarize their work concerning the mechanism of pancreatic lipase activation. The activation of lipase by submicellar SDS concentrations was found to imitate closely enough its activation by an interface. Lipase activation was shown to be caused by changes in the rate constants for substrate chemical transformation and to involve conformational changes of the enzyme and its association. The complex of a conformationally modified lipase with the detergent, which acts as a 'structure-forming' agent, is associated with native lipase molecules setting up their active site. The mechanism of lipase activation at an interface both in vitro and in vivo is discussed.