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V A Fried

Publications and source records attributed to V A Fried.

52 records · Page 3Linked to original sources
PubMed1987

Two BALB/c anti-arsonate idiotype families: two heavy chain variable regions (Vh) shared with anti-DNP antibodies are used by one family while a Vh similar to anti-GAT antibodies is used by the other.

Amino terminal amino acid sequences were obtained for both the heavy (H) and light (L) chains of seven BALB/c anti-arsonate (Ar) monoclonal antibodies representing the 5AF6 and 3C6 idiotype (id) families described in this strain. 5AF6 family H chains showed strong homology to the germ-line gene sequence for the A strain 36-60 family. However, four to five identical H chain sequence differences for two of these antibodies (5AF6 and 95B5), as well as two previously reported related sequences (92D5, 94B10), suggested they were encoded by a different Vh. The 36-60 family Vh genes have been shown to be identical to the Vh gene of the anti-DNP binding myeloma M460 [Dzierzak et al., J. Immun. 136, 1864-1870 (1986)]. H chains amino acid sequences derived from an id-460+ anti-DNP hybridoma and a germ-line gene differing from the 30-60-like Vh sequence [Dzierzak et al., J. Immun. 136, 1864-1870 (1986)] were found to be virtually identical to the 95B5 and 5AF6 Vh sequences. This suggests that the same two related H chains making up two subsets of the 5AF6 anti-Ar id family are also both used in two subsets of the id-460 anti-DNP response. 5AF6 family L chains were highly homologous to the other Vk2 L chains of the 36-60 family. 3C6 family H chains can be placed in the Vh l group, are unrelated to the described anti-Ar H chain families, and have been placed in a new anti-Ar Vh family, Ars-E. The 3C6 H is similar, however, to a Vh used by a BALB/c anti-GAT idiotype family of antibodies. 3C6 L chains were of the murine kappa chain group, Vk8 and most resembled an L chain from an A strain monoclonal anti-Ar having no defined idiotype.

Amino Acid Sequence↗
PubMed1987

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Based on nucleotide sequence analysis of the hemagglutinin (HA) gene from the virulent and avirulent A/chicken/Pennsylvania/83 influenza viruses, it was previously postulated that acquisition of virulence was associated with a point mutation that resulted in loss of a glycosylation site. Since there are two potential glycosylation sites in this region of the HA molecule and since all Asn-Xaa-Thr/Ser sequences in the HAs of different strains are not necessarily glycosylated, the question remained open as to whether either one of these sites was glycosylated. We now provide direct evidence that a site-specific glycosylation affects cleavage of the influenza virus HA and thus virulence. We have identified the glycosylation sites on the HA1 subunit from the virulent and avirulent strains by direct structural analysis of the isolated proteins. Our results show that the only difference in glycosylation between the HA1s of the virulent and avirulent strains is the lack of an asparagine-linked carbohydrate on the virulent HA1 polypeptide at residue 11. Further, we show that the HA1s of both the avirulent and virulent viruses are not glycosylated at one potential site, while all other sites contain carbohydrate. Amino acid sequence analysis of the HA1 of an avirulent revertant of the virulent strain confirmed these findings.

Amino Acid Sequence↗
PubMed1987

Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation.

Ubiquitin is a protein of 76 amino acids found in every eukaryotic cell. Although ubiquitin is implicated in ATP-dependent nonlysosomal protein degradation and is also conjugated to specific cellular proteins, the role played by ubiquitin in cellular events has not been defined. We report that purified ubiquitin has intrinsic proteolytic activity and demonstrate that this activity is comparable to that of other well-characterized proteases. Monoclonal antibodies specific to ubiquitin inhibit proteolysis. Ubiquitin has protease activity over a broad pH range with an optimum at pH 8.0. It is stimulated by Ca2+ and is inhibited by high concentrations of phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. Ubiquitin will cleave proteins at a limited number of sites. We propose that the ubiquitination of a protein can convert that protein into an ad hoc specific protease and models are presented as to how this can play a role in regulating a variety of cellular events.

Antibodies, Monoclonal↗
PubMed1986

Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched-chain glycoprotein.

Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino termini, one of which corresponds precisely to the amino terminus of ubiquitin. This observation extends the province of this conserved polypeptide to the cell surface and leads to a proposed model of the receptor complex as a core polypeptide modified by glycosylation and ubiquitination. Independent antibodies to ubiquitin serve to identify additional cell surface species, an indication that ubiquitination of cell surface proteins may be more general. It is proposed that functional binding of lymphocytes to lymph node high endothelial venules might involve the ubiquitinated region of the receptor; if true, cell surface ubiquitin could play a more general role in cell-cell interaction and adhesion.

Amino Acid Sequence↗
PubMed1986

Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species.

The lymphocyte cell surface receptor for the high endothelial venules (HEV's) of peripheral lymph nodes is specifically recognized by the monoclonal antibody MEL-14. Three independent complementary DNA (cDNA) clones, each of which encodes the protein ubiquitin, were detected by virtue of the expression of the MEL-14 antigenic determinant on cDNA-beta-galactosidase bacterial fusion proteins. The antigenic determinant defined by MEL-14 resides in the carboxyl terminal 13-amino-acid proteolytic peptide of ubiquitin, but is undetected in intact undenatured ubiquitin and other cellular ubiquitinated proteins. Antisera and monoclonal antibodies to ubiquitin determinants bind to the surface of both HEV-receptor positive and negative cell lines. The MEL-14-identified cDNA clones hydridize to RNA transcripts that encode tandemly repeated ubiquitins. Sequence analysis of these polyubiquitin cDNA's does not identify a leader sequence for export to the cell surface. The expression of the MEL-14 epitope of ubiquitin depends upon its local environment. The steady-state levels of expression of the ubiquitin messenger RNA's do not correlate with either the tissue derivation of the RNA or the expression of the lymphocyte HEV receptor. Regulation of the expression of the HEV receptor is not likely to reflect the transcriptional control of ubiquitin genes, but rather to reflect control of the expression of the HEV core polypeptide or its level or form of ubiquitination.

Amino Acid Sequence↗
PubMed1986

The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences.

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.

Amino Acid Sequence↗
PubMed1985

Protein quantitation at the picomole level: an O-phthaldialdehyde-preTSK column-derivatization assay.

A fluorescent protein assay was described wherein an isocratic high-performance liquid chromatography system was used to separate the o-phthaldialdehyde-derivatized proteins from interfering components. Using a small TSK guard column equilibrated in 0.1% sodium dodecyl sulfate, it was demonstrated that all proteins and peptides examined, containing more than 22 residues, coelute in the excluded volume and were resolved from fluorescent signals contributed by commonly used reagents. The assay was linear over a useful range of 3 ng to 1 microgram of protein and required less than 15 microliter of sample.

Aldehydes↗
PubMed1983

A novel proteolytic activity apparently initiating degradation of beta-galactosidase nonsense fragments in in vitro extracts of Escherichia coli.

Our previous in vivo studies demonstrated that large premature fragments of beta-galactosidase are degraded in Escherichia coli by a common pathway, and the initial event appears to be a site-specific cleavage (McKnight, J. L., and Fried, V. A. (1981) J. Biol. Chem. 256, 9652-9661). We now have developed a cell-free system that retains the specificity of this early cleavage event. Immunochemical techniques were used to isolate and quantitate the polypeptide substrate and products in pulse-chase experiments. The in vitro system has an activity that quantitatively converts the prematurely terminated A polypeptide of the lacZ non-sense mutant CSH-10 to the 90-kilodalton common B polypeptide intermediate observed in vivo. The activity is localized in the cytoplasm since the cleavage reaction is not affected by osmotic shock of whole cells or removal of the membrane fraction from cell-free extracts. The lon mutation capR9, which blocks this degradation pathway in vivo, does not affect the initial cleavage event in cell-free extracts of CSH-10 carrying this mutation. The in vitro cleavage event in extracts of lon+ CSH-10 or the isogenic lon- mutant is not stimulated by addition of ATP, not inhibited by depletion of ATP pools by hexokinase-2-deoxyglucose treatment, and not inhibited by EDTA or phenylmethylsulfonyl fluoride. These results suggest that the ATP-dependent proteolytic activity of the lon gene product does not directly catalyze this primary cleavage event.

Escherichia coli↗
PubMed1981

Limited proteolysis. Early steps in the processing of large premature termination fragments of beta-galactosidase in Escherichia coli.

The mechanism by which large premature termination fragments of beta-galactosidase were degraded in Escherichia coli was studied using quantitative immunoprecipitation techniques. Two different lacZ nonsense mutants which produced apparent primary translation products of 96,000 and 109,000 daltons, respectively, were both shown to produce a second beta-galactosidase-related polypeptide of Mr = 90,000. These 90,000-dalton polypeptides appeared to be the same in both strains since they co-migrated when analyzed as a mixture on sodium dodecyl sulfate-polyacrylamide gels and were indistinguishable when analyzed by one-dimensional peptide mapping. Pulse-chase experiments established a stoichiometric precursor-product relationship between the primary mutant gene products (called the A polypeptides) and the common 90,000-dalton polypeptide (called the B polypeptide). No intermediates were detected between the A and B polypeptides. We propose that there is a common pathway for the degradation of these different large fragments of beta-galactosidase. According to this model, the first step would be a specific endoproteolytic cleavage of the primary translation product which produces the 90,000-dalton polypeptide as a common intermediate. The kinetic analysis demonstrated a first order decay of both A and B polypeptides but, surprisingly, the first order rate constant for the decay of A appeared dependent upon the induction regimen. This result suggested that degradation may possibly be autoregulated either by the intracellular level of A or by other intermediates in the degradation pathway.

Antigen-Antibody Complex↗
PubMed1981

Membrane biogenesis. Evidence that a soluble chimeric polypeptide can serve as a precursor of a mutant lac permease in Escherichia coli.

A mutant in the Escherichia coli lac permease, called Yf, appears to be defective in the biogenesis and proper assembly of this membrane protein. It was proposed that this defect led to the accumulation of a precursor of the mutant permease (Fried, V. A. (1977) J. Mol Biol. 114, 477-490). In this communication, evidence is presented that the lacYf mutant accumulates a novel lac-specific soluble polypeptide with a molecular weight of 87,000. Detected by double-label analysis on sodium dodecyl sulfate gels, and identified as a lac-specific polypeptide on a two-dimensional gel system, this polypeptide is immunoprecipitated by anti-transacetylase antibody. Pulse-chase experiments are consistent with the hypothesis that it is converted in vivo into a lac-specific membrane protein with an apparent molecular weight of 28,000, which appears to be the mutant lac permease. The results suggest that the 87,000-dalton soluble protein is a precursor of the mutant lac permease. It is proposed that this precursor is a polyprotein chimera containing both the lacY and lacA gene products.

Cell Membrane↗
PubMed1980

lac Thiogalactoside transacetylase of Escherichia coli K-12 and ML.

The lac thiogalactoside transacetylase was purified from both a wild-type Escherichia coli K-12 strain (H3000) and an E. coli ML strain (ML308). These enzymes are indistinguishable by using several criteria. The subunit molecular weight of the enzyme is 24,800, which is significantly less than the previously reported value of 30,000. Although the function of the thiogalactoside transacetylase is unknown, it is suggested that this enzyme plays an important role in lactose utilization since its structure and enzymatic activity have been conserved.

Acetyltransferases↗
PubMed1978

Interactions between lipopolysaccharide and phosphatidylethanolamine in molecular monolayers.

Lipopolysaccharide and phosphatidylethanolamine are the two major lipid constituents of the membrane of Salmonella typhimurium. Interactions between the purified lipopolysaccharide and phosphatidylethanolamine were studied in molecular monolayers at air-water interfaces. The equilibrium surface pressures of mixed films of lipopolysaccharide and phosphatidylethanolamine were determined as a function of the film composition. The plot of the equilibrium surface pressrue vs. the area occupied by phosphatidylethanolamine molecules exhibited two distinct regions. Below a phosphatidylethanolamine surface concentration at which 55% of the surface was occupied by phosphatidylethanolamine molecules, the equilibrium pressure was invariant and had the value of a pure lipopolysaccharide monolayer at maximum compression. At phosphatidylethanolamine surface concentrations in excess of 55% surface area occupation (phosphatidylethanolamine/lipopolysaccharide (mol/mol) greater than 16), the equilibrium surface pressure was a function of the surface concentration of phosphatidylethanolamine. The results suggest a simple model in which lipopolysaccharide and phosphatidylethanolamine form a complex in which each lipopolysaccharide molecule is surrounded ("lipidated") by a shell of approx. 16 phosphatidylethanolamine molecules.

Lipopolysaccharides↗
PubMed1973

Organic solvents as probes for the structure and function of the bacterial membrane: effects of ethanol on the wild type and an ethanol-resistant mutant of Escherichia coli K-12.

The effects of ethanol on the growth of a wild-type Escherichia coli K-12 are described. These effects include a reduction of the steady-state growth rate and an interference with the division process. They appear as an immediate response to the addition of ethanol and are rapidly reversed by removal of ethanol. Mutants were selected that could grow at a concentration of ethanol that stopped wild-type growth. The growth of one of the mutants we studied (strain S9L100) is stimulated by the presence of ethanol, methanol, or dimethyl sulfoxide. This strain exhibits pleiotropic growth defects including abnormal cell division and morphology. It also appears to have an altered lac permease function which is not due to a mutation in the Y gene itself. We conclude that this mutant has an altered membrane and that the membrane defect may be the cause of the abnormal growth properties. The use of compounds which serve as general membrane perturbants and mutants resistant to these perturbants form a system accessible to both genetic and physical-biochemical techniques.

Bacterial Proteins↗
PubMedDate not supplied

Insulin-like growth factor II receptor as a multifunctional binding protein.

The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.

Amino Acid Sequence↗