The kallikrein, kininase and related peptides activities in central Asian snake venoms.

The quantitative content estimation of kininogenases, kininases and related peptides have been made for Central Asian snake venoms: V. lebetina turanica and E. multisquamatus (gen. Vipera and Echis, fam. Viperidae), Ag. halys halys (gen. Agkistrodon, fam. Crotalidae) and N. oxiana (gen. Naja, fam Elapidae). It has been demonstrated, that all venoms investigated cause the contractile effect, when acting on isolated smooth muscle preparations. Kinin-like contractile activity was found in the low molecular weight fraction of the cobra venom. This action has the prolonged character as compared with bradykinin, but apart from it, results in the inactivation of the rat uterus because of cytotoxic components presence. The specific bradykinin-potentiating effect of the low molecular weight fraction of the E. multisquamatus venom has been discovered. It has been found, that the effect is connected with inhibition of the kininase II (angiotensin I converting enzyme, ACE). Two peptide inhibitors was isolated and characterized from this fraction.

[Structural-functional characteristics of vasoactive peptides from the Vespa orientalis venom]. / Strukturno-funktsional'naia khrakteristika vazoaktivnykh peptidov iz iada shershnia Vespa orientalis.

Two vasoactive peptides are isolated from the Vespa orientalis venom by gel filtration and ion-exchange chromatography. The physicochemical and functional properties of peptides are studied. Vasoactive peptides show the myotropic activity and hypotensive action which is of prolonged character as compared with bradykinin. Their complete amino acidic sequences are determined. One of the peptides is a similar structural analogue of bradykinin.

Identification of ceruloplasmin messenger RNA sequences in heterogeneous nuclear RNA from rat liver.

The distribution of the sequences of ceruloplasmin mRNA in different fractions of heterogeneous nuclear RNA from rat liver was studied using cDNA transcripts of highly purified mRNA as hybridization probe. The content of ceruloplasmin mRNA sequences in poly(A)-containing and poly(A)-free subfractions of heterogeneous nuclear RNA is respectively 1 and 27 molecules per a hepatocyte. Heterogeneous nuclear RNA carrying the sequences of ceruloplasmin mRNA sedimented in sucrose gradients containing formamide, as a broad zone around the 56S peak. Denaturing electrophoresis followed by the transfer of RNA onto diabenzyloxymethyl paper and hybridization with [32P]-cDNA revealed multiple high molecular weight fractions of ceruloplasmin pre = mRNA (9.0, 6.6, 2.2 and 1.6 megadaltons) in the non-adenylated fraction of nuclear RNA and a single 1.1-1.2 megadalton zone in poly(A)-containing nuclear RNA, the latter being equal in size to the mature ceruloplasmin mRNA from liver polysomes.

[Structural organization of rat ceruloplasmin gene]. / Strukturnaia organizatsiia gena tseruloplazmina krysy.

Distribution of ceruloplasmin-coding sequences among fragments of rat nuclear DNA obtained after complete cleavage with seven restriction endonucleases was studied using highly specific complementary DNA probes. Three different procedures were used for the synthesis of cDNAs, whose relative advantages and disadvantages are discussed in this work. The number of restriction fragments carrying ceruloplasmin gene sequences varied from two to five depending on the enzyme used. The total molecular weight of these fragments was several times higher than the minimal length of ceruloplasmin structural gene deduced from the molecular size of mRNA. The restriction endonuclease cleavage of the partial double-stranded transcript of ceruloplasmin mRNA coding for about 60% of its length from the 3'-end has shown that distribution of restriction endonuclease cleavage sites on the dsDNA differs in the position of cleavage sites on the ceruloplasmin gene in cellular DNA. Hybridization of cDNA with total cellular DNA allows to determine 1.3-1.4 copies of ceruloplasmin gene in the rat haploid genome. Proceeding from the data obtained it may be stated that the rat ceruloplasmin gene is constructed of several structural gene segments cut by introns.

[Nuclear precursors for ceruloplasmin-coding messenger RNA from rat liver]. / Iadernye predshestvenniki informatsionnoi RNK, kodiruiushchei tseruloplazmin, iz pecheni krysy.

The distribution of the sequences coding for ceruloplasmin (CP) in rat liver heterogeneous nuclear RNA (hnRNA) was studied using highly specific CP cDNA as a hybridization probe. The content of CP-coding sequences in poly(A)-containing and poly(A)-free subfractions of hnRNA was shown to be respectively 1 and 27 equivalents of CP mRNA molecule per one hepatocyte. The gel electrophoresis of hnRNA under strongly denaturing conditions with the subsequent transfer of RNA to diazobenzyloxymethyl paper and hybridization with [32P]-cDNA probe showed that CP mRNA sequences were of multiple molecular weight distribution. In particular, 9.0, 6.6, 2.4 and 1.6 megadalton fractions of non-polyadenylate hnRNA carried CP-coding sequences while the only hand that hybridized to CP cDNA was detected in polyadenylated hnRNA. This band was of a molecular weight 1.1-1.2 megadaltons corresponding to that of cytoplasmic CP mRNA. The hybridization of high molecular weight hnRNA with full-length CP cDNA followed by the determination of the size of cDNA fragments protected against SI nuclease demonstrated that coding sequences of CP pre-mRNA are interrupted by intervening sequences.

Highly purified ceruloplasmin messenger RNA from rat liver. Physico-chemical and functional characteristics.

Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 . 10(6) daltons which is large enough to code for a putative precursor of ceruloplasmin (approximately 700 amino acid acids). Ceruloplasmin mRNA contains 3'-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5'-end of ceruloplasmin mRNA is blocked with confronting m7G residue which is a component of cap I (m7G5'ppp5'XmpAp). The addition of ceruloplasmin mRNA to wheat-germ cell free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.

Intracellular distribution of rat-liver polyribosomes synthesizing coeruloplasmin.

Three independent approaches (binding of 125I-antibodies to polysomes, cell-free translation of polyadenylated mRNA in a wheat germ system and hybridization of RNA with a specific complementary DNA probe) were used to study the intracellular distribution of polysomes involved in the synthesis of coeruloplasmin as well as of coeruloplasmin mRNA sequences in rat liver. It was shown that only membrane-bound polysomes contain both nascent chains of coeruloplasmin and coeruloplasmin mRNA sequences. The size of coeruloplasmin polysomes is 16-18 monomers/mRNA molecule and their proportion is about 0.4-0.6% of total liver polysomes. The proportion of coeruloplasmin mRNA is 0.0025% of the total polysomal RNA and 0.29% of poly(A)-containing mRNA. These values correspond to coeruloplasmin mRNA concentration about 400-535 molecules per parenchymatous liver cell, 90% of those mRNA molecules were recovered from polysomes while the remaining 10% were from postpolysomal supernatant.

Complex molecular structure of the gene coding for rat ceruloplasmin.

The distribution of ceruloplasmin-coding sequences among the fragments of rat nuclear DNA obtained after the complete digestion with seven restriction endonucleases (EcoRI, BamHI, BspI, HindIII, KpnI, BglII and XhoI) was studied using highly specific cDNA probes. Although only a single copy of this gene per rat haploid genome was detected in DNA-cDNA hybridization in solution, the number of restriction fragments carrying the sequences of ceruloplasmin (CP) gene varied from two to five, depending upon the enzyme used, and their total length was several times higher than the minimal length of CP-coding gene, as deduced from the size of mRNA (2.3 Md for double-stranded DNA). The partial double stranded DNA transcript of ceruloplasmin mRNA coding for about 70% of its length (from 3'-end) does not contain recognition sites for some restriction endonucleases generating multiple fragments of CP gene in cellular DNA. These data are consistent with a split pattern of CP gene which seems to consist of several exons and introns. The partial protection from S1 nuclease of discrete fragments of full-length cDNA after annealing with high molecular weight nuclear RNA is consistent with this assumption and seems to be an indication that exons and introns are joined into a functional unit coding for high mol wt. CP pre-mRNA.

[Enzymatic synthesis and characterization of DNA complementary to ceruloplasmin mRNA from rat liver]. / Fermentativnyi sintez i kharakteristika DNK, komplementarnoi tseruloplazminovoi mRNK iz pecheni krysy.

Poly(A) containing rat liver 21S RNA homogeneous in polyacrylamide gel electrophoresis under denaturing conditions and stimulating the synthesis of ceruloplasmin in a cell-free proteinsynthesizing system, was used as a template for reverse transcription in the presence of T10 primer and highly purified reverse transcriptase from avian myeloblastosis virus. The cDNA made this way was characterized by means of hybridization kinetics with mRNA, by melting of the hybrids formed and by chain length measurements. To increase the degree of representativity, the ceruloplasmin mRNA was fragmented by mild alkaline treatment, enzymatically polyadenylated and transcribed. The cDNA made was fully characterized and the kinetic complexity measured by hybridization with the mRNA was found to be equal to 2300 nucleotides as compared with the value of 3000 nucleotides is expected from gel electrophoresis data. The observed difference may indicate the presence of repeated sequences in the given mRNA. The sufficient representativitness of the synthesized cDNA and its specificity with respect to ceruloplasmin mRNA allows to use it as a molecular probe to study the ceruloplasmin gene structure.

[Physico-chemical characteristics of highly purified ceruloplasmin mRNA from rat liver]. / Fiziko-khimicheskaia kharakteristika vysokoochishchennoi tseruloplazminovoi mRNK iz pecheni krysy.

Highly purified preparations of mRNA coding for ceruloplasmin (CP) ere isolated from rat liver polyribosomes using indirect immunoprecipitation of CP polysomes and poly(U)-sepharose chromatography of polysomal RNA. The homogeneity of CP mRNA was as high as 86--90%. The molecular weight of CP mRNA is 1.3 . 10(6) daltons which is in excess when compared to the minimal size of mRNA necessary to code for CP precursor (about 700 amino acid residues). The base composition of CP mRNA is of AU-type. The experiments on end-labeling with [3H]borohydride after periodate oxidation whowed that CP mRNA contains 3'-terminal poly(A). Poly(U)-sepharose chromatography with stepwise temperature elution revealed length heterogeneity of poly(A) consisting of particular, different thermal subfractions of CP mRNA contain poly(A) consisting of 38, 90 and 165 adenylate residue. 5'-end of CP mRNA is block with inverted 7-methylguanosine (m7G) which is reducible with [3H]borohydride after periodate oxidation. This m7G residue is a component of RNAse- and alkali-resistant oligonucleotide, which structure according to net charge value and its shifts after various enzymatic treatments, is m7G5'ppp5'XmpAp.