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R Shapira

Publications and source records attributed to R Shapira.

At least 55 records · Page 3Linked to original sources
PubMed1986

Structure and activities of a variant ubiquitin sequence from bakers' yeast.

Ubiquitin is an extremely conserved protein, with an identical sequence throughout the animal kingdom. However, the gene sequence of the yeast protein [Ozkaynak, E., Finley, D., & Varshavsky, A. (1984) Nature (London) 312, 663-666] predicts three amino acid differences. This implies that some functions or binding interactions of ubiquitin are different in yeast and animal cells. In an effort to define these differences, ubiquitin has been purified to homogeneity from bakers' yeast and characterized. Amino acid analysis of the protein and the isolated tryptic peptides confirms the primary structure of this protein as predicted from the gene sequence. This result indicates that the gene sequenced is the transcriptionally active gene from yeast. The conformation of yeast ubiquitin is similar to human ubiquitin as judged by circular dichroism, sensitivity to trypsin, and Stokes radius. Yeast and animal ubiquitins show identical activities in supporting ubiquitin-dependent protein degradation and in the ATP-pyrophosphate exchange reaction catalyzed by the purified ubiquitin-adenylating enzyme. Thus, the three conservative amino acid differences between yeast and animal ubiquitins have very little effect on the structure of ubiquitin or its activity in the ubiquitin-dependent proteolytic system. These results suggest that at least some of the evolutionary pressure preventing sequence variation among animal ubiquitins stems from one or more of its nonproteolytic functions.

Amino Acid Sequence↗
PubMed1986

Multiple myeloma in Northern Israel, 1970-1979.

From 1970 to 1979, multiple myeloma was diagnosed in 184 residents of Northern Israel. The data were analyzed for variations by demographic factors, ethnicity, country of origin, and urban/rural residence areas. The average annual age-adjusted incidence rate per 100,000 residents was 1.78. The rates for males and females were 2.1 and 1.46/10(5) person-years, respectively. The male to female incidence ratio was 1.44:1.0. The average incidence rate was significantly higher for Jews than for Arabs and was higher for European Jews than for Afro-Asians or native Israelis. Urban areas had significantly higher rates than rural areas. A statistically significant increase in the incidence rates was demonstrated for the years 1975 to 1979 compared to the period 1970 to 1974. The increase was evident in all population categories, but was particularly prominent in females, the Jewish population, urban areas, and in the oldest age group. The results of the current study suggest that both culturally derived factors and industrial environment may play a role in the etiology of multiple myeloma.

Adult↗
PubMed1986

Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography.

The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms.

Amino Acid Sequence↗
PubMed1985

The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis.

A novel cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis system which is useful for the separation of native forms of proteins consistent with their molecular weights is reported here. Many proteins examined in this system demonstrated the same association patterns which have been shown by other techniques to exist under nondenaturing conditions. In addition, biological activity could be assayed directly in the gel after electrophoresis. Based on the peculiar characteristics of cetyltrimethylammonium bromide, a possible explanation which may account for the behavior of proteins in this system is presented.

Cetrimonium↗
PubMed1983

Myelin proteins: degradation in rat brain initiated by metabolites causative of maple syrup urine disease.

Maple syrup urine disease (MSUD), an inborn error of metabolism in humans, is expressed as an inability to oxidatively decarboxylate the branched-chain alpha-keto acids derived from leucine, isoleucine and valine. Rats 14 days old were injected intracranially with a solution containing leucine, alpha-ketoisocaproate, and tracer amounts of 3H-lysine. Myelin isolated from these rat brains at 28 days of age had a washed dry weight 85 per cent of controls. The protein content of the myelin prepared from treated and control rats was identical, as were the specific activities of the individual proteins separated by polyacrylamide gel electrophoresis. Myelin protein from treated rats was deficient in myelin high molecular weight proteins including glycoproteins, and degradation products of these proteins were observed in myelin of treated rats. MSUD associated metabolites in man may initiate a process leading to the proteolytic degradation of myelin proteins, thereby producing abnormal myelin sheaths.

Animals↗
PubMed1983

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils.

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.

Adult↗
PubMed1983

Multiple forms of myeloperoxidase from human neutrophilic granulocytes: evidence for differences in compartmentalization, enzymatic activity, and subunit structure.

Multiple forms of myeloperoxidase from normal human neutrophilic granulocytes obtained from a single donor can be resolved by carboxymethyl (CM)-cellulose ion-exchange column chromatography into three forms (I, II, and III) designated in order of elution of adsorbed enzyme using a linear salt gradient. Selective solubilization of individual forms of the enzyme by detergent (form I) or high-ionic-strength procedures (forms II and III) suggested that these forms of the enzyme were compartmentalized differently. All three forms were purified by a combination of preferential extraction, manipulation of ionic strength, and ion-exchange and molecular sieve chromatography. Purified forms II and III had similar specific activities for a variety of substrates. Form I was less active toward several of these same substrates, most notably iodide, with a specific activity about one-half that of forms II and III. All forms had similar spectral properties characteristic of a type alpha heme. The amino acid compositions of the three forms were similar, yet significant differences were found in selected residues such as the charged amino acids. Native polyacrylamide gel electrophoresis resolved small differences in mobility between the forms which were consistent with the charge heterogeneity observed on CM-cellulose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis data were consistent with the generally accepted subunit structure of two heavy chains and two light chains. All three forms contained a small-molecular-weight subunit of Mr 11,500. Form I contained a large subunit of Mr 63,000, while forms II and III contained a corresponding subunit of Mr approximately 57,500. We conclude that heterogeneity of human myeloperoxidase is accompanied by differences in cellular compartmentalization, enzymatic activity, and subunit structure.

Amino Acids↗
PubMed1983

Encephalitogenic activity of the small form of mouse myelin basic protein in the SJL/J mouse.

Mouse myelin basic proteins (MBP) were prepared by ion-exchange chromatography of an acid extract of homogenized, delipidated mouse brain. The use of a linear salt gradient during ion-exchange chromatography gave three peaks, 1, 2, and 3, of MBP. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of these peaks showed that peaks 2 and 3 were a mixture of the large and small forms of mouse MBP, whereas peak 1 was solely the small form. Tryptic peptides of mouse and rat small form MBP were compared by high-performance liquid chromatography. The elution times and absorbances at 210 nm were identical for 16 of 21 peaks derived from small form MBP of the two species. Amino acid analysis showed that 10 corresponding peaks from the small form of mouse and rat MBP had identical amino acid composition. When tested for encephalitogenicity in SJL/J mice, both the mixture of large and small forms of mouse MBP and the small form mouse MBP were highly encephalitogenic.

Amino Acid Sequence↗
PubMed1982

Isolation characterization and idiotype of Lewis rat antibodies against peptide 68-88 of guinea pig myelin basic protein.

Antibodies against the 19 amino acid encephalitogenic peptide )residues 68-88) of guinea pig myelin basic protein (GPBP) were raised in Lewis (Le) rats. Anti-peptide antibodies were isolated from immune ascitic fluids by affinity chromatography using peptide 43-88-Sepharose 4B. The purified antibodies were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing. Immunoglobulin class was determined by radioimmunoassay. Anti-idiotypic (anti-ID) antibodies were raised in a rabbit using purified anti-peptide antibodies from a single rat. The results of these experiments showed antibody heterogeneity both within an individual anti-peptide antiserum and between antisera from different rats. Antibody activity was found in IgG1, IgG2, and IgE immunoglobulin classes. Isoelectric focusing revealed multiple bands within a population of purified antibodies with significant pattern variation from one antiserum to another. Idiotypic characterization showed various levels of cross-reactive idiotypes present in some sera while these were absent in others.

Animals↗
PubMed1981

Turnover of myelin proteins of rat brain, determined in fractions separated by sedimentation in a continuous sucrose gradient.

Rats that received intracranial injections of [3H]leucine at 14 days of age were killed on days 17, 24, 38, 55, and 89 post-injection. Brains were homogenized and the myelin membranes separated in a sucrose density gradient. At day 17 sodium dodecylsulfate polyacrylamide gels of water-shocked, delipidated membrane fractions showed a difference in the specific activity of myelin proteins across the gradient. A decrease in specific activity was found in all of the proteins in the denser fractions, compared with the lighter fractions. As time after injection progressed, the difference became more pronounced; a two- to threefold decrease in specific activity was seen across the gradient in the various myelin proteins. The proteins of the lightest membrane fractions retained their high specific activity throughout the experiment in spite of extensive new myelin synthesis. Taking this new myelin into account, the decrease in specific activity in the denser myelin fractions could be explained by isotope dilution. Therefore, proteins present in at least some of the myelin are essentially stable.

Aging↗
PubMed1976

Basis of microheterogeneity of myelin basic protein.

The basic protein of bovine central nervous system myelin contains a single polypeptide chain of 170 amino acids. Multiple components of basic protein have been observed on disc gel electrophoresis and ion exchange chromatography at alkaline pH, but the basis of the microheterogeneity has not been established. In the present study myelin basic protein from bovine spinal cord was chromatographed on carboxymethylcellulose at pH 10.4 in glycine buffer/2 M urea. Three major peaks were obtained, identified as components 4, 5, and 6 in the oder of their elution from the column by a linear salt gradient. The amino acid compositions of tryptic peptides from components 4 and 6 were identical and the COOH-terminal sequence, Ala-Arg-Arg, was intact for all three components. Component 4 was found to differ from component 6 by partial phosphorylation of threonine 98 and serine 165. This modification was estimated to account for 50% of component 4. Component 5 differed from component 6 by partial deamidation of glutamine residues 103 and 147, which accounted for 80% of this component. These modified glutamine residues were also present in component 4 and constituted another 15% of this component. It was considered that component 6 was the native, unmodified species of basic protein and that component 4 differed by a net negative charge of 2, and component 5 by a net negative charge of.1 as a result of these modifications. The nonrandom nature of the modifications suggested the involvement of specific enzymes.

Amino Acids↗