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.
Different Protein Patterns in Normal and Mastitic Milks as Revealed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis