Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.     

Regulation of glutamate dehydrogenase in Bacillus subtilis.

The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium. The highest specific activity for this enzyme was found when B. subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source. It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.     

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.     

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.     

Pseudomonas aeruginosa ExoS ADP-ribosyltransferase inhibits ERM phosphorylation

Pseudomonas aeruginosa causes life-threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96-219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234-453) encodes a 14-3-3-dependent ADP-ribosyltransferase domain which transfers ADP-ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high-affinity substrates for ADP-ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP-ribosyltransferase activity. MALDI-MS and site-directed mutagenesis studies determined that ExoS ADP-ribosylated moesin at three C-terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP-ribosylated-moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP-ribosylation directly inhibited ERM phosphorylation. Expression of dominant active-moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP-ribosylation. These data explain how the expression of the ADP-ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.     

Quantifying raft proteins in neonatal mouse brain by 'tube-gel' protein digestion label-free shotgun proteomics

Background  

The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. For accurate analyses of the raft proteome, simplified protocols are needed to avoid excessive sample handling and purification steps.     

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.     

Amino acid sequence of P-57, a neurospecific calmodulin-binding protein

The amino acid sequence was determined for bovine brain P-57, a neurospecific, membrane-associated, calmodulin-binding protein. It consists of a single 239-residue polypeptide chain blocked at its amino terminus and containing an unusually hydrophilic amino acid composition. Seventy percent of the molecule is composed of Glu/Gln, Ala, Lys, Asp/Asn, and Pro; there is only one aromatic residue. A lack of favorable cleavage sites required that a particularly wide variety of digests and subdigests be performed to obtain appropriate sets of overlapping peptides. This protein is clearly homologous with the cDNA-derived sequence of mouse brain P-57, although the bovine protein is 12 amino acid residues longer; the homology is less obvious in the middle sections of the two sequences. Bovine brain P-57 lacks homology with any other protein in an updated sequence database. A segment reported to interact with calmodulin (Arg-Gly-His-Ile-Thr-Arg-Lys-Lys-Leu) is placed at residues 43-51 within the only extended segment of P-57 that carries the net positive charge that would favor that interaction. There is no hydrophobic segment characteristic of many proteins that interact with membranes.     

Characterization of cell-envelope fractions of chemotrophically and phototrophically grown Rhodospirillum tenue

Envelope preparations of chemotrophically and phototrophically grown Rhodospirillum tenue were isolated and characterized on the basis of their contents and composition in phospholipids and fatty acids, as well as on the basis of their enzyme activities, absorption spectra and polypeptide patterns. Both preparations were similar in their contents in phospholipids and fatty acids. Their total fatty acids were characterized by a rather high percentage of saturated fatty acids. The activities of the respiratory reactions were considerably higher in chemotrophic than in phototrophic membranes. This is true especially for activities of the terminal oxidase which were over 20 times greater. The affinities of the corresponding enzymes to their respective substrates (K _m ) differed with differences in the culture conditions. Under chemotrophic conditions the K _m values for the NADH-dependent reactions were lower than those values under phototrophic conditions, whereas the K _m values for the succinate dependent reactions were higher. The low temperature (77°K) near infrared spectrum of the phototrophic membrane showed a peak at 875 nm which was not detectable in the chemotrophic membrane. The polypeptide patterns, in the presence of sodium dodecyl sulfate, of both preparations were quite similar except for the presence of two low molecular weight proteins (M. Wt. 12,000 and 10,000) in the phototrophic membrane which were absent in the chemotrophic membrane. Both envelope preparations were further fractionated into enriched cytoplasmic membrane and outer membrane fractions which were identified on the basis of their contents in 2-keto-3-deoxyoctonate, ubiquinone 8 and bacteriochlorophyll (in the case of the phototrophic membrane) and their enzyme activities. The buoyant densities of the corresponding fractions from both envelope preparations were found to be equal. The data presented in this paper demonstrate that envelope preparations of chemotrophically and phototrophically grown R. tenue are similar in their contents in 2-keto-3-deoxyoctonate, ubiquinone 8, phospholipids and fatty acids, yet differ significantly in their spectra, protein patterns and enzyme activities.Abbreviations BChl bacteriochlorophyll - UQ_s ubiquinone 8 - KDO 2-keto-3-deoxyoctonate - PG phosphatidyl glycerol - PE phosphatidyl ethanolamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - SDS sodium dodecyl sulfate