Characterization and partial purification of parathyroid hormone messenger RNA

L. Stolarsky, B. Kemper

Research output: Contribution to journalArticlepeer-review

Abstract

The messenger RNA coding for parathyroid hormone has been partially purified from bovine parathyroid tissue. After fractionation of the RNA by sucrose gradient centrifugation, affinity chromatography on oligo(dT)-cellulose or poly(U)-Sepharose, and formamide-sucrose gradient centrifugation, specific translational activity is enriched 175-fold for parathyroid hormone mRNA. The mRNA is about 50% pure at this stage as estimated by electrophoretic analysis of 125I-labeled RNA on acrylamide gels. The 125I-RNA appears to be about 90% pure after additional fractionation by electrophoresis on 98% formamide-acrylamide gels. The primary translational product of all fractions of the RNA is pre-proparathyroid hormone which represents 40% of the total protein synthesized as analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis. About 75 and 65% of the [35S]methionine-labeled translational products of the formamide-sucrose and formamide-gel RNA fractions, respectively, are precipitated by antiserum against bovine parathyroid hormone. The molecular weight of parathyroid hormone mRNA is dependent on the method used to estimate it. Molecular weights of about 200,000 are obtained by sucrose gradients centrifugation in the presence or absence of 50% formamide, 258,000 by acrylamide gel electrophoresis in gels containing 98% formamide, and 330,000 by gel electrophoresis in nondenaturing gels. The average size of the poly(A) sequence at the 3' terminus is 60 bases long. A 7-methylguanosine cap is probably present at the 5' terminus since 7-methylguanosine 5'-phosphate inhibits the translation of the mRNA. The 530 to 700 bases present in parathyroid hormone mRNA excluding the poly(A) sequence are considerably more than the 345 bases required to synthesize pre-proparathyroid hormone.

Original languageEnglish (US)
Pages (from-to)7194-7201
Number of pages8
JournalJournal of Biological Chemistry
Volume253
Issue number20
StatePublished - 1978

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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