Search

An important function of influenza virus neuraminidase (NA) is the removal of sialic acid residues from virion components in order to prevent the aggregation of virus particles. In previous communications we have reported that reassortant viruses containing the NA gene of A/USSR/90/77 (H1N1) virus and HA genes of H3, H4, H10, or H13 subtypes had a tendency to virion aggregation at 4 degrees C and that the virion clusters irreversibly dissociated after the treatment with bacterial neuraminidase. It was concluded that in such reassortants the removal of sialic acid residues is inefficient. Nonaggregating variants of the reassortants were selected in the course of serial passages in embryonated chicken eggs. In the present paper a reassortant virus, R2, having the HA gene of A/Duck/Ukraine/1/63 (H3N8) virus and the other genes of A/USSR/90/77 (H1N1) virus, as well as its non-aggregating passage variants and both parent viruses, have been studied in order to reveal the presence of unremoved sialic acid residues in the virions. An assay of sialic acid content by high-performance liquid chromatography with fluorescent detection has revealed the presence of sialic acid in the purified virus preparations of A/USSR/90/77 (H1N1) virus and the R2 reassortant and its nonaggregating variants, whereas only trace amounts of sialic acid have been detected in the A/Duck/Ukraine/1/63 (H3N8) parent virus. The data obtained with the use of the labeled "indicator" virus suggest that the unremoved sialic acid residues are present at the virion surface. The nonaggregating variants have been shown to possess a lower affinity toward high-molecular-weight sialic acid-containing substrates compared to the initial reassortant R2. Sequencing of HA genes has revealed amino acid changes in the nonaggregating variants compared to the initial reassortant. One substitution, N248D in HA1, is the same in two independently selected nonaggregating variants. The presented data suggest that the complete removal of sialic acid residues by viral NA from the virion components is not obligatory for the absence of virus particle aggregation: the latter may be achieved (in the reassortants and, presumably, in the wild-type virus) through a balance between the degree of HA affinity toward the sialic acid-containing receptors and the extent of the removal of sialic acid residues by NA.

Active preparations of Na+,K(+)-ATPase containing three types of catalytic isoforms were isolated from the bovine brain to study the structure and function of the sodium pump. Na+,K(+)-ATPase from the brain grey matter was found to have a biphasic kinetics with respect to ouabain inhibition and to consist of a set of isozymes with subunit composition of alpha 1 beta 1, alpha 2 beta m and alpha 3 beta m (where m = 1 and/or 2). The alpha 1 beta 1 form clearly dominated. For the first time, glycosylation of the beta 1-subunit of the alpha 1 beta 1-type isozymes isolated from the kidney and brain was shown to be different. Na+,K(+)-ATPase from the brain stem and axolemma consisted mainly of a mixture of alpha 2 beta 1 and alpha 3 beta 1 isozymes having identical ouabain inhibition constants. In epithelial and arterial smooth muscle cells, where the plasma membrane is divided into functionally and biochemically distinct domains, the polarized distribution of Na+,K(+)-ATPase is maintained through interactions with the membrane cytoskeleton proteins ankyrin and spectrin (Nelson and Hammerton, 1989; Lee et al., 1996). We were the first to show the presence of the cytoskeleton protein tubulin (beta 5-isoform) and glyceraldehyde-3-phosphate dehydrogenase in a high-molecular-weight complex with Na+,K(+)-ATPase in brain stem neuron cells containing alpha 2 beta 1 and alpha 3 beta 1 isozymes. Consequently, the influence of not only subunit composition, but also of glycan and cytoskeleton structures and other plasma membrane-associated proteins on the functional properties of Na+,K(+)-ATPase isozymes is evident.

A homogeneous metalloproteinase has been isolated with a 28% yield from the culture fluid of Bacillus mesentericus, strain B-313. The isolation procedure included chromatography on bacitracin-silochrome and gel filtration on Acrylex P-10 and Sephadex G-75. The enzyme has a molecular mass of 41,000 Da; its N-terminal sequence, which appears as A-A-T-T-G-T-G-T-T-L-K-G-K-T-V-S-L-N-I, is identical with that of the B. amyloliquefaciens enzyme. Like other metalloproteinases, the enzyme is inhibited by o-phenanthroline and EDTA, has an activity maximum at 55 degrees C and pH 6.5-7.2, is stable at pH 7.0-9.5 and at temperature below 45 degrees C for several hours, and is irreversibly inactivated in acid media. As can be judged from the kcat/Km ratio dependence on pH, two ionogenic groups with pKa of 7.4 and 6.2 are involved in the catalytic act, presumably the imidazol group of histidine and the carboxylic group. Within synthetic peptides the enzyme hydrolyzes the bonds formed by the amino group of hydrophobic amino acids, mostly of leucine residues.

Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B. megaterium strain 599. The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined. The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+. The enzyme has a peak activity at 60-65 degrees C. The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours. In acid media the enzyme undergoes irreversible inactivation. The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine. The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.

The gene of human mutant (serine-17) fibroblast interferon was isolated with the use of highly efficient oligonucleotide-directed mutagenesis. On the basis of the constructed expression plasmid pPR-IFN Ser17 a strain producing human mutant beta-interferon (VKPM V-4678) was developed. It was shown that the specific activity of the human mutant (serine-17) fibroblast interferon was 1 order of magnitude higher than that of the recombinant interferon which reaches the specific activity of natural fibroblast interferon.

A serine proteinase having an activity optimum at pH 6.7-8.2 has been isolated from amylorisine P-10x (a mixture of Aspergillus oryzae enzymes) by chromatography on DEAE-Sephadex A-50 and bacitracin Sepharose 4B. The proteinase is fully inactivated by phenylmethylsulfonylfluoride and diisopropylfluorophosphonate, the specific inhibitors of the enzyme, and has a pI at pH 7.5. The molecular mass of serine proteinase is 30000 Da; its amino acid composition appears as: Met2, Asp33, Thr18, Ser29, Glu21, Pro9, Glu32, Ala38, Val24, Ile16, Leu15, Tyr8, Phe8, His8, Lys18, Arg4, Trp6. The N-terminal sequence of the serine proteinase: Gly-Leu-Thr-Thr-Gln-Lys-Ser-Ala-Pro-Trp-Gly-Leu-Gly-Ser-Ile-Ser-Xaa-Lys- Gly-Gln-Gln-Ser-Thr-Asp-Tyr-Ile-Tyr, which coincides practically completely with the corresponding sequence of alkaline proteinase of A. oryzae, ATCC20386, has been determined. Similar to subtilisin, the enzyme catalyzes the condensation of leucine and alanine p-nitroanilides with N-benzyloxycarbonyl-alanyl-alanine and glycyl-alanine methyl esters.

Prochymosin can be converted into chymosin by an action of external proteinases. Thus, thermolysin at pH 5.05 converts calf prochymosin into active Phe-chymosin, which is one amino acid longer than chymosin from the N-terminus with a yield of 73%. Even better results were achieved with prochymosin activation by Legionella pneumophila metalloproteinase. Apparently the stretch of prochymosin polypeptide chain adjacent to the normally observed activation point becomes available for an attack by an external proteinase at pH 5.0-6.0. These data indicate that the intermolecular activation pathway might be of physiological importance.

The tryptic peptide sequences of the N-terminal domain ("true toxin") of delta-endotoxin of Bac. thuringiensis subspecies alesti carrying 282 amino acid residues were determined. A comparison of these sequences with the primary structures of delta-endotoxin of subspecies kurstaki (K-1, K-73) determined by an analysis of corresponding structural genes revealed a conservative region of "true toxin" (residues 29-346) and a hypervariable region (residues 347-617) carrying multiple (not less than 50%) substituents of amino acid residues. It is essential that the amino acid substituents in the variable region are distributed unevenly, being grouped into several highly variable sites carrying 7 to 31 residues. Besides, tryptic peptides of subspecies alesti delta-endotoxin were found to contain peptides having no homologs in the structures of subspecies kurstaki delta-endotoxins. It seems probable that such an uneven distribution of amino acid substituents in the structures of delta-endotoxins of subspecies alesti and kurstaki reflects the functional differences in the two halves of the N-terminal domain ("true toxin"), one of which (i. e., conservative) may be responsible for the toxic effect, while the other one (i. e., variable) seems to participate in toxin interactions with the appropriate receptors of larvae gut.

Carboxypeptidase T, an extracellular carboxypeptidase from Thermoactinomyces sp. was isolated and purified by affinity chromatography on bacitracin adsorbents. The enzyme homogeneity was established by SDS electrophoresis (Mr = 38 000) and isoelectrofocusing in PAAG (pI 5.3). Carboxypeptidase T reveals a mixed specificity in comparison with pancreatic carboxypeptidases A and B and cleaves with nearly the same efficiency the peptide bonds formed by the C-terminal residues of basic and neutral hydrophobic amino acids. The enzyme is insensitive to serine and thiol proteinase inhibitors but is completely inhibited by EDTA and o-phenanthroline. The maximal enzyme activity is observed at pH 7-8. With an increase of temperature from 20 to 70 degrees C the enzyme activity is enhanced approximately 10-fold. In the presence of 1 mM Ca2+ the enzyme thermostability is also increased. In terms of some properties, e.g. substrate specificity carboxypeptidase T is similar to metallocarboxypeptidase secreted by Streptomyces griseus. The N-terminal sequence of carboxypeptidase T: Asp-Phe-Pro-Ser-Tyr-Asp-Ser-Gly- Tyr-His-Asn-Tyr-Asn-Glu-Met-Val-Asn-Lys-Ile-Asn-Thr-Val-Ala-Ser-Asn-Tyr- Pro-Asn - Ile-Val-Lys-Thr-Phe-Ser-Ile-Gly-Lys-Val-Tyr-Glu-Gly-Xaa-Gly-Leu- coincides by 21% with that of pancreatic carboxypeptidases A and B. Thus, it may be concluded that these enzymes originate from a common precursor.

Large peptide fragments of human leucocyte interferon-alpha 2 (INF-alpha 2) were obtained by limited proteolysis with trypsin, pepsin, thermolysine and Bacillus amyloliquefaciens intracellular serine proteinase. The ability of the fragments to bind murine monoclonal antibodies NK2 raised against INF-alpha 2 was studied by the immunoblotting technique. The region of sequence 110-149 is the most sensitive to proteolytic attack, being probably exposed on the surface of the INF-alpha 2 molecule. INF-alpha 2 fragments 1-139, 1-147, 1-149 are capable of binding antibodies, whereas fragments 1-109 and 1-112 do not bind antibodies NK2. A comparison of the primary structure of human leucocyte and murine leucocyte INF families in the region of sequence 110-139 and an analysis of the ability of human INF differing in amino acid sequences to bind antibodies NK2 demonstrated that the antigenic determinant for antibodies NK2 is the sequence Glu114-Asp115-Ser116-Ile117 of the INF-alpha 2 molecule.

Extracellular beta-galactosidase from P. canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration. The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5. In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium. The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined.

The thermo- and acid-stable trypsin, chymotrypsin and intracellular proteinases inhibitor (TAS-inhibitor) from rabbit serum was digested by trypsin, and its domain (Mr 6200) with antitryptic activity was obtained in homogeneous state. The N-terminal amino acid sequence of this domain was established by automatic Edman degradation: Thr-Val-Ala-Ala-Cys-Asx-Leu-Pro-Ile-Val-Pro-Gly-Pro-X-Arg-Gly-Ile-Phe-X- Leu-X-Ala-Phe-X-Ala-Val-X-Gly. A high degree of homology of the primary structures rabbit, human and bovine TAS-inhibitors was demonstrated.