Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

Heat shock response in Neurospora crassa: purification and some properties of HSP 80

The heat shock response of Neurospora crassa was investigated. A 80-kilodalton heat shock protein (HSP 80) was purified to near homogeneity from heat-shocked mycelial extracts employing ammonium sulphate fractionation, gel filtration, and ion-exchange and affinity chromatography. It was observed to migrate as a single band on one-dimensional sodium dodecyl sulphate--polyacrylamide gels, with a molecular mass of approximately 83 kilodaltons (kDa). On two-dimensional gels it resolved into four polypeptide species with isoelectric points in the acidic range, which on staining with periodic acid--Schiff method were demonstrated to be glycosylated. In the native state, HSP 80 had a molecular size of approximately 610 kDa.     

CsgA, an extracellular protein essential for Myxococcus xanthus development.

CsgA mutants of Myxococcus xanthus appear to be defective in producing an extracellular molecule essential for the developmental behaviors of this bacterium. The csgA gene encodes a 17.7-kilodalton polypeptide whose function and cellular location were investigated with immunological probes. Large quantities of the CsgA gene product were obtained from a lacZ-csgA translational gene fusion expressed in Escherichia coli. The chimeric 21-kilodalton protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified polyclonal antibodies raised against the fusion protein were used to determine the cellular location of the native CsgA protein by colloidal gold labeling and transmission electron microscopy. Between 1,100 and 2,200 extracellular molecules of CsgA per developing M. xanthus cell were detected, most of which were associated with the extracellular matrix. The anti-CsgA antibodies inhibited wild-type development unless they were first neutralized with the fusion protein. Together these results suggest that the CsgA gene product has an essential, extracellular function during development, possibly as a pheromone.     

Protein Differences between Two Isogenic Cultivars of Barley (Hordeum vulgare L.) that Differ in Sensitivity to Photoperiod and Far-Red Light

A photoperiodically sensitive cultivar of barley (Hordeum vulgare L. Shabet) (BMDR-8) and an isogenic, single-gene recessive mutant of this genotype that is insensitive to photoperiod (BMDR-1) were grown under continuous cool white light with or without supplemental far-red fluorescent light. BMDR-1 initiates flowers 6 days after germination, irrespective of light treatment, whereas BMDR-8 remains vegetative for at least a week longer, even in continuous light. When far-red light is added, the delay of flowering in BMDR-8 is overcome and both genotypes initiate floral primordia at the same time. Total phenol extracted proteins of seedlings of both genotypes were resolved by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No protein differences were found between the genotypes when isoelectric focusing gels were run in the first dimension. Two qualitative genotypic differences were found when nonequilibrium pH gradient gel electrophoresis was run in the first dimension. An 85-kilodalton polypeptide (A) and a 26-kilodalton polypeptide (B) were always present in BMDR-8 but never found in BMDR-1. The levels of A appeared to decrease from the BMDR-8 during the first 3 days of far-red treatment but did not disappear completely even after 6 days of growth in the presence of farred. Polypeptide B decreases rapidly in continuous cool white light but is stabilized by far-red. The phytochrome content of BMDR-1 was found to be greater than that for BMDR-8. This increase appears to be caused by the type I (etiolated-tissue abundant) phytochrome pool, even in plants grown in continuous light.     

Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins.

The polypeptide composition of highly purified densonucleosis virus was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viral proteins showed a different behavior in sodium dodecyl sulfate-gels in comparison with the marker proteins. Therefore, the molecular weights were estimated by analyzing the retardation of the electrophoretic mobility of these proteins in gels with increasing polyacrylamide concentrations. Four structural proteins with molecular weights of 49,000, 58,500, 69,000, and 98,000 were found, ant they were designated p49, p59, p69, and p98, respectively. There are several indications that p98 is a dimer of p49. The relative quantity of the structural proteins in a virion suggests that at least p49 (accounting for +/-70% of total protein mass) is a capsid protein and that there will be 12 capsomers per virion.     

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high-molecular-weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS-polyacrylamide gels. The apparent molecular weight of the center of the band varies from ～230,000 on 13% acrylamide gels to ～130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.     

Carbohydrate composition and electrophoretic properties of tomato polygalacturonase isoenzymes

Two polygalacturonase isoenzymes, PG I and PG II, were extracted from Murrieta tomato and purified by gel exclusion and ion-exchange chromatography. The kinetic constants and activation energies of the purified isoenzymes have been determined. Polygalacturonase I has two polypeptide chains (Mr = 47 500 and 41 400) whereas polygalacturonase II is a single polypeptide (Mr = 47 500) as shown by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate. Both isoenzymes are glycoproteins. Through gas liquid chromatography, polygalacturonase II was shown to contain 4.6% neutral hexoses and 1.5% amino sugars. There are eight D-mannose, two L-fucose, two D-xylose and three N-acetylglucosamine residues per mole of PG II. The carbohydrate portion of PG II was shown to be attached to the protein part through an N-acetylglucosaminylasparaginyl bond.     

Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography.

We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.     

Purification of leucoplast pyruvate kinase from developing castor bean endosperm

Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases.     

Isolation and characterization of an extremely basic protein from adenovirus type 5.

By starch-gel electrophoresis and a staining method that is highly sensitive for argininyl residues, adenovirus type 5 was found to contain two minor basic polypeptides of extreme cathodic mobility in addition to the two known core proteins. The fastest-migrating polypeptide, named mu protein, and the second fastest polypeptide are found in adenovirions and virus-infected KB cells but not in top components or in uninfected cells. The top components and infected cells contain an additional basic polypeptide, presumably P-VII, that migrates slightly slower than polypeptide VII. None of the basic polypeptides of adenovirions was electrophoretically identical to the host histone. The basic proteins of adenovirions were purified by urea phosphocellulose column chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two minor basic core proteins, mu and another component, have similar mobilities in sodium dodecyl sulfate-polyacrylamide gels as a complex of polypeptides X-XII. After further purification on a Sephadex G-75 column, the mu protein was found to have a molecular weight of about 4,000. Amino acid analysis showed that the mu protein lacks tryptophan and 69% of the total amino acid residues are basic, that is, 54% arginine, 13% histidine, and 2% lysine. Only eight amino acids seem to contribute to make the mu polypeptide. There are 125 copies of the mu polypeptide per 1,000 copies of polypeptide VII in a virion.     

Purification and properties of an endothelial cell growth factor from human platelets

An endothelial cell growth factor has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the factor consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about 20 ng/ml (440 pM). Heparin did not potentiate the activity, nor did the factor bind to heparin immobilized on Sepharose. The purified factor was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin fibroblasts. Chromatofocusing revealed that the pI of the factor was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelial cell mitogens with affinities for heparin.     

Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.     

Chromoplast-Specific Proteins in Capsicum annuum

Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast-specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M_r of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species.     

Resolution of two subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.     

Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.     

Immunochemical demonstration of the iron-sulfur protein in Complex III of mitochondrial electron-transfer chain

An iron-sulfur protein of Complex III was purified by phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography. The purified preparation was homogeneous as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a specific antibody directed against this protein was raised in a rabbit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunoperoxidase reaction indicated that Complex III possesses a single polypeptide which reacts with the antibody. It was also found that the iron-sulfur center-containing subunits identified so far in Complex I did not cross-react with the antibody, indicating that they are antigenically unrelated to the iron-sulfur protein of Complex III.     

The identification of myosin in rabbit hepatocytes.

A myosin-like protein was identified in isolated rabbit liver cells. It was extracted with high-ionic-strength buffer containing ATP, and purified by gel filtration in the presence of iodide. The myosin polypeptide was indistinguishable in size from the heavy chain of muscle myosin as determined by electrophoresis on polyacrylamide gels and gel filtration in the presence of sodium dodecyl sulfate. The hepatic myosin had an amino acid composition similar to that of muscle myosin, but lacked 3-methylhistidine. The Mg2+ -ATPase of the myosin was not activated by muscle actin. At low ionic strength, in the presence of Mg2+, the protein aggregated to form bipolar filaments 0.3 mum in length. A protein which resembled muscle actin in size and amino acid composition was extracted along with the myosin. Based on scans of stained sodium dodecyl sulfate polyacrylamide gels, the myosin content was estimated as 0.3% to 0.4% of the cell protein. The actin-like component was present in approximately ten-fold excess by weight. This ratio suggests that the organization and function of myosin in the hepatocyte is very different from that in the muscle cell.     

Purification of terminal riboadenylate transferase from calf thymus gland.

A poly(A) polymerase has been purified from the soluble protein fraction of calf thymus gland. The activity is cytoplasmic and nonparticulate. Mn-2+ATP is the preferred substrate. On the basis of disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels, gel filtration, and sedimentation velocity in sucrose gradients, the enzyme has a molecular weight of 62,000 and appears to consist of one polypeptide chain. The enzyme preparation is shown to be nearly homogeneous by disc gel electrophoresis and isoelectric-focusing. The activity has a pI of about 7.4. The specific activity of the enzyme is about 1700 mumol per hour per mg of protein, giving a turnover number of about 1800 mol of substrate per mol of enzyme min- minus 1. The activity is highly specific for ATP and is inhibited by other ribonucleoside triphosphates. It is sensitive to high levels of RNA-polymerase inhibitors. Km for oligoadenylate is 50 muM in the presence of Mn-2+ and 200 muM in Mg-2+ and equivalent Vmax is achieved with either metal ion. The initiator function may be filled by a variety of oligoribonucleotides having a free 3'-OH.