scholarly journals Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are regulated by reversible phosphorylation and/or Ca2+ ions

1997 ◽  
Vol 325 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Pauline DOUGLAS ◽  
Emmanuelle PIGAGLIO ◽  
Albert FERRER ◽  
Nigel G. HALFORD ◽  
Carol MacKINTOSH
Keyword(s):  
Nitrate Reductase ◽  
Molecular Mass ◽  
Phosphorylation Site ◽  
Gel Filtration ◽  
Leaf Extracts ◽  
Peptide Substrates ◽  
Spinacea Oleracea ◽  
Spinach Leaf ◽  
Cnbr Cleavage ◽  
Coa Reductase

In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis(Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. All three kinases gave phosphopeptide CNBr-cleavage maps of HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506–513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PKI is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-independent, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of ∼140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognised a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported previously for the SNF1-like enzyme, HRK-A. Our results indicate a considerable complexity of kinase cascades mediating the regulation of assimilatory and biosynthetic pathways in response to environmental stimuli in plants.

scholarly journals Purification of a Necrosis-Inducing, Host-Specific Protein Toxin from Spore Germination Fluid of Alternaria panax

2003 ◽  
Vol 93 (3) ◽  
pp. 323-328 ◽  
Author(s):  
H. A. Quayyum ◽  
M. Gijzen ◽  
J. A. Traquair
Keyword(s):  
Molecular Mass ◽  
Spore Germination ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
American Ginseng ◽  
Leaf Extracts ◽  
Water Extracts ◽  
Protein Toxin ◽  
Alternaria Panax

Spore germination fluid of Alternaria panax, the causal agent of Alternaria blight of American ginseng (Panax quinquefolius), collected from water droplets or aqueous ginseng leaf extracts produced visible water-soaked lesions on wounded, detached leaflets after incubation for 48 h. Maximum development of brown, necrotic spots occurred 4 to 5 days after inoculation on attached and detached ginseng leaflets. Of 15 plant species tested, only American ginseng was susceptible to applications of spore inoculum or spore germination fluid. The phytotoxic activity of the spore germination fluid was destroyed by heat and treatment with proteinase A. The phytotoxic factor was retained by an ultrafiltration membrane with a 30-kDa molecular mass cut-off. Purification of the phytotoxic protein, named AP-toxin, was performed by anion exchange and gel filtration chromatography. Bioactive fractions eluted as a single peak. By comparison with protein standards, a molecular mass of 35 kDa was estimated for the native protein. The denatured protein toxin also had a mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Production of the protein toxin was induced on American ginseng leaflets and water extracts of ginseng leaves but not on leaves of other nonhost plants and their water extracts. The results show that A. panax produces a host-specific, proteinaceous toxin during colonization and pathogenesis of ginseng leaves.

Identification of Ser-543 as the Major Regulatory Phosphorylation Site in Spinach Leaf Nitrate Reductase

1996 ◽  
Vol 8 (3) ◽  
pp. 505 ◽  
Author(s):  
Markus Bachmann ◽  
Naomasa Shiraishi ◽  
Wilbur H. Campbell ◽  
Byung-Chun Yoo ◽  
Alice C. Harmon ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Phosphorylation Site ◽  
Regulatory Phosphorylation ◽  
Spinach Leaf ◽  
Leaf Nitrate

scholarly journals Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

1996 ◽  
Vol 8 (3) ◽  
pp. 505-517 ◽  
Author(s):  
M Bachmann ◽  
N Shiraishi ◽  
W H Campbell ◽  
B C Yoo ◽  
A C Harmon ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Phosphorylation Site ◽  
Regulatory Phosphorylation ◽  
Spinach Leaf ◽  
Leaf Nitrate

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

1991 ◽  
Vol 278 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A J Rivett ◽  
S T Sweeney
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Rat Tissues ◽  
Specific Antibodies ◽  
Multicatalytic Proteinase ◽  
Cell Extracts ◽  
Liver And Kidney ◽  
Lysosomal Proteinase

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

scholarly journals Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni

1988 ◽  
Vol 254 (2) ◽  
pp. 419-426 ◽  
Author(s):  
P M Wiest ◽  
E J Tisdale ◽  
W L Roberts ◽  
T L Rosenberry ◽  
A A F Mahmoud ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Free Amino ◽  
Protein Modification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Reductive Methylation ◽  
Amide Linkage

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.

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