scholarly journals Isolation of a bifunctional domain from the pentafunctional arom enzyme complex of Neurospora crassa

1983 ◽  
Vol 213 (2) ◽  
pp. 405-415 ◽  
Author(s):  
D D S Smith ◽  
J R Coggins
Keyword(s):  
Neurospora Crassa ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Polyacrylamide Gel ◽  
Enzyme Complex ◽  
Two Dimensional ◽  
Shikimate Dehydrogenase ◽  
Activity Staining

Limited proteolysis of the arom enzyme complex of Neurospora crassa by trypsin or subtilisin yielded a stable fragment of Mr 68000. This fragment, which was purified by two-dimensional polyacrylamide-gel electrophoresis, was shown by activity staining to contain the shikimate dehydrogenase active site, and by substrate labelling with 3-dehydroquinate and NaB3H4 to contain the 3-dehydroquinase active site. The fragment thus constitutes a bifunctional domain containing the two enzymic activities that are known, from genetic evidence, to be located adjacently at the C-terminal end of the pentafunctional arom polypeptide.

Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme

1982 ◽  
Vol 2 (8) ◽  
pp. 993-1001
Author(s):  
D Wang ◽  
Y Furuichi ◽  
A J Shatkin
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Hela Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Enzyme Complex ◽  
Cell Nuclei ◽  
Dodecyl Sulfate ◽  
Enzyme Intermediate

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.

Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins

1998 ◽  
Vol 19 (5) ◽  
pp. 797-801 ◽  
Author(s):  
Michele Mortarino ◽  
Gabriella Tedeschi ◽  
Armando Negri ◽  
Fabrizio Ceciliani ◽  
Luciano Gottardi ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Seminal Plasma Proteins
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