Use of stem proteins and isozymes for the identification of celery varieties

Separation of stem proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis permitted classification of 17 celery varieties (Apium graveolens L.) into 7 groups. Several of these groups could be further subdivided into 11 subgroups with gel electrophoresis patterns of 4 isozyme systems. Classification from gel banding patterns was consistent with that based on pedigree histories.Abbreviations KD kilodaltons - SDS-PAGE sodium dodecyl sulfate polyacrylamide electrophoresis - BSA bovine serum albumin     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Anomalous behavior of goldfish IgM heavy chain in sodium dodecylsulfate polyacrylamide gel electrophoresis

By sodium dodecylsulfate polyacrylamide gel electrophoresis, the heavy chain of the serum immunoglobulin (IgM) of the goldfish (Carassius auratus) differs not only from other studied vertebrate serum IgM heavy chains, but also from other vertebrate lymphocyte membrane IgM heavy chains including those from the goldfish itself. This difference, an increase in apparent Mr of approximately 5000, was investigated by assessing in comparison with the IgM heavy chain of human and rainbow trout (Salmo gairdneri) the following properties: (1) molecular size by gel filtration in denaturing buffers; (2) carbohydrate content, by direct analysis; (3) intrinsic net charge, by isoelectric focusing; (4) net hydrophobicity, deduced from amino acid analysis; and (5) sodium dodecylsulfate binding by direct measurement. Results indicate that goldfish IgM heavy chain is indistinguishable from other IgM heavy chains in terms of (a) its gel-filtration behavior in denaturing conditions, (b) its carbohydrate content (which is similar to trout IgM heavy chain) and (c) its intrinsic net charge and hydrophobicity. However, goldfish IgM does differ from the other proteins studied in its detergent-binding ability and it is this behavior that is concluded to be the cause of its unusual mobility in sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia californica

The biosynthesis and processing of low molecular weight protein (presumed neurosecretory protein) in cells R15, R14 and L11 of Aplysia californica was studied at high resolution by polyacrylamide slab gel electrophoresis in sodium dodecylsulfate. The number of low molecular weight proteins detected in each cell ranges from 3 in R14 and L11 to 5 or 6 in R15. In each of the cells studied, the low molecular weight protein consists of a primary precursor of ca. 12,000 daltons, and its proteolytic processing products. In each cell, the smallest protein, or in the case of R14, one of the two smallest proteins, accumulates to a significant extent, suggesting that it might correspond to a final processed neurohormone. In cell R15, the biosynthesis of the primary precursor and its subsequent processing to smaller peptides is largely unaffected by removal of extracellular calcium, by replacement of calcium with cobalt or by inhibition of spontaneous bursting via stimulation of the brachial nerve.     

The stress-70 protein family in diplopods: induction and characterization

To induce stress-70 proteins (hsp70), adults of the millipede Julus scandinavius (Diplopoda) were exposed to leaf litter contaminated with different concentrations of Cd²⁺ (10, 30, 50 and 60 mg·kg^-1 as CdCl₂). The expression of hsp70 was investigated by semiquantitative and qualitative biochemical methods. After SDS-gel electrophoresis and Western blotting a subsequent digital image analysis showed that increasing dietary concentrations of Cd²⁺ resulted in elevated levels of hsp70, which in turn indicated proteotoxic condition. Qualitative results were obtained by two-dimensional gel electrophoresis. A stress-70 protein family, similar to that of other arthropods, was detected in Julus scandinavius: at least five different proteins with an approximate molecular weight of 68, 69, 70, 77, and 78 kDa could be distinguished after heat shock as well as after Cd²⁺ exposure.Abbreviations IEF isoelectric focusing - hsp heat shock protein(s) - grp glucose regulated protein(s) - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.     

Azido auxins : photoaffinity labeling of auxin-binding proteins in maize coleoptile with tritiated 5-azidoindole-3-acetic Acid

Tritiated 5-azidoindole-3-acetic acid (5-N₃-[7-³H]IAA), a photoaffinity labeling agent, was used to photolabel proteins of a crude microsomal preparation from maize (Zea mays L., Bear Hybrid, WF9 × BR38) coleoptile. Approximately 50% of the bound radioactivity was solubilized in 5 molar urea containing Triton X-100, and the extract was fractionated using a variety of techniques. High performance liquid chromatography demonstrated that, although many membrane proteins incorporated tritiated label, only a few showed reduced incorporation in the presence of excess indole-3-acetic acid. By contrast, no detectable reduction in incorporation was observed in the presence of excess naphthalene-1-acetic acid. Results from isoelectric focusing gel electrophoresis indicate that the proteins that showed reduced incorporation of photolyzed 5-N₃-[7-³H]IAA in the presence of IAA fell into two main groups: one which focuses between pH 5.2 and 5.7 (pI 4.8-5.3) and another around pH 6.2 (pI 5.8). In sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins migrated as four bands with apparent molecular weights of 60, 49, 45, and 37 kilodaltons. The auxin-transport inhibitor, 2,3,5-triiodobenzoic acid, competes for the labeling by 5-N₃-[7-³H]IAA, suggesting that some of these proteins may be involved in auxin transport.     

Identification of novel protein targets regulated by maternal dietary fatty acid composition in neonatal rat liver

Polyunsaturated fatty acids regulate metabolic pathways, which in early development could have important consequences to adaptation to extra-uterine life and programming of metabolic pathways. Female rats were fed one of two diets identical in all nutrients, except that the fat in one diet was high in unsaturated fatty acids (UFA) and the other low UFA, through gestation and lactation. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis of protein extracts from 3-day old pup liver resolved over 800 proteins. Employing MALDI-TOF MS and peptide mapping we identified 11 proteins that differed more than three-fold between the groups, 10 up regulated and one down regulated in the high UFA group. The up-regulated proteins included fructose-1,6-bisphosphatase 1, glycerol-3-phosphate dehydrogenase, galactokinase 1, 40S ribosomal protein SA, elongation factor 1-gamma, protein disulfide-isomerase A6, catalase, cytokeratin-8 and 60 kDa heat shock protein, and the down-regulated protein was argininosuccinate synthase, none having been previously reported to be regulated by fatty acids in the developing liver. We further determined that fructose-1,6-biphosphatase is acetylated at the N-terminus. We demonstrate that early fatty acid nutrition impacts hepatic metabolic pathways relevant to gluconeogenesis, redox balance and nitric oxide signaling.     

Identification of novel protein targets regulated by maternal dietary fatty acid composition in neonatal rat liver

Polyunsaturated fatty acids regulate metabolic pathways, which in early development could have important consequences to adaptation to extra-uterine life and programming of metabolic pathways. Female rats were fed one of two diets identical in all nutrients, except that the fat in one diet was high in unsaturated fatty acids (UFA) and the other low UFA, through gestation and lactation. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis of protein extracts from 3-day old pup liver resolved over 800 proteins. Employing MALDI-TOF MS and peptide mapping we identified 11 proteins that differed more than three-fold between the groups, 10 up regulated and one down regulated in the high UFA group. The up-regulated proteins included fructose-1,6-bisphosphatase 1, glycerol-3-phosphate dehydrogenase, galactokinase 1, 40S ribosomal protein SA, elongation factor 1-gamma, protein disulfide-isomerase A6, catalase, cytokeratin-8 and 60 kDa heat shock protein, and the down-regulated protein was argininosuccinate synthase, none having been previously reported to be regulated by fatty acids in the developing liver. We further determined that fructose-1,6-biphosphatase is acetylated at the N-terminus. We demonstrate that early fatty acid nutrition impacts hepatic metabolic pathways relevant to gluconeogenesis, redox balance and nitric oxide signaling.     

Fractionation of some bovine whey proteins by recycling gel filtration on a large scale.

Fractionation of bovine whey concentrate was performed by gel filtration on Sephadex G-75 both on a laboratory scale and on a large scale. By a recycling procedure and improved separation was obtained and the whey proteins were resolved into four fractions in the weight ratio 3:12:1:4. The fractions were analysed by polyacrylamide gel (PAG) electrophoresis and the apparent molecular weights were determined by thin layer gel chromatography (TLG) and by sodium dodecylsulfate (SDS) gel electrophoresis.     

Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia californica.

The biosynthesis and processing of low molecular weight protein (presumed neurosecretory protein) in cells R15, R14 and L11 of Aplysia californica was studied at high resolution by polyacrylamide slab gel electrophoresis in sodium dodecylsulfate. The number of low molecular weight proteins detected in each cell ranges from 3 in R14 and L11 to 5 to 6 in R15. In each of the cells studied, the low molecular weight protein consists of a primary precursor of ca. 12,000 daltons, and its proteolytic processing products. In each cell, the smallest protein, or in the case of R14, one of the two smallest proteins, accumulates to a significant extent, suggesting that it might correspond to a final processed neurohormone. In cell R15, the biosynthesis of the primary precursor and its subsequent processing to smaller peptides is largely unaffected by removal of extracellular calcium, by replacement of calcium with cobalt or by inhibition of spontaneous bursting via stimulation of the brachial nerve.     

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

Purification and properties of 3-hexulosephosphate synthase from Methylomonas M 15.

3-Hexulosephosphate synthase, the first enzyme of the ribulose monophosphate cycle, was purified 15-fold from methanol-grown Methylomonas M 15. The purification procedure involved chromatography on DEAE-cellulose, Sephadex G-75, and DEAE-Sephadex A-50. The purified enzyme was more than 95% pure as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was calculated to be 43000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a molecular weight of 22000. The enzyme catalyzes specifically the condensation formaldehyde with ribulose 5-phosphate to yield D-arabino-3-hexulose 6-phosphate. The Km values were found to be 1.1 mM for formaldehyde and 1.6 mM for ribulose 5-phosphate. A bivalent cation is essential for activity and stability of the enzyme, Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5--8.0.     

Occurrence of cytochrome c-554 in a facultative methylotroph,Protaminobacter ruber strain NR-1, during bacteriochlorophyll formation

A facultative methylotroph, Protaminobacter ruber was grown under two different conditions (aerobically grown under light, and aerobically in the dark after a light period). Bacteriochlorophyll was synthesized inducibly in the cells which were initially grown in the ligt and then grown in the dark, while bacteriochlorophyll was not found in the cells cultured under continuous light. Cytochrome c-554 was solely synthesized parallel to bacteriochlorophyll after switching from light to dark conditions. Both cytochrome c-554 and bacteriochlorophyll levels in the membrane preparation reached to a plateau in 24 h after switching from light and dark conditions. This cytochrome was membrane-bound and its M _r was 45,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis. The midpoint potential was 358 mV at pH 7. Other major membrane-bound cytochromes and two soluble cytochromes were present in both types of cells and their content did not change irrespective of growth conditions.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Bchl bacteriochlorophyll     

Studies of the gene expression in a somatic hybrid of dihaploid Solanum tuberosum and diploid S. brevidens using two-dimensional protein electrophoresis

Two-dimensional protein electrophoretic patterns of leaf, stem and microtuber were compared between a somatic hybrid (Solanum tuberosum + S. brevidens) and parental plants. Polypeptide spots observed in leaf of the somatic hybrid (BT-1) were similar to those of S. brevidens. In the stem of BT-1, the spots characteristic for each parental plants were also observed. Three specific spots (W, X, Y) found in BT-1 were identical to those of S. tuberosum, however their appearance in S. brevidens depended on the culture conditions (observed at 16h daylength regime, but not in the dark with high sucrose concentration). Potato tuber storage protein patatin was observed in small amounts in the microtubers of BT-1. The data indicated that gene expression unique to each parental plants also existed in the somatic hybrid.Abbreviations BAP 6-benzylaminopurine - 2,4-D (2,4-dichlorophenoxy) acetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Affinity labeling of rat-kidney gamma-glutamyl transpeptidase.

The reaction of gamma-glutamyl transpeptidase from rat kidney with a glutamine analog, 6-diazo-5-oxo-L-norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half-maximum rate of inactivation was 6 mMat pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active-site-directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6-diazo-5-oxo-L-norleucine. Inactivation of the enzyme with 6-diazo-5-oxo-L-[6=14C]norleucine as well as with 6-diazo-5-oxo-L[1,2,3,4,5-14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label/248000 g enzyme protein. A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C-labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.     

Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes

Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (32P)orthophosphate and incubating isolated membrane with (gamma-32P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with Mr 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.