Tyrosine hydroxylase: purification from PC-12 cells, characterization and production of antibodies

Donald M. Kuhn, Melvin L. Billingsley

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

Tyrosine hydroxylase has been purified to homogeneity from cultured PC-12 cells. The protein migrates as a single band with a molecular weight of 60,000 on sodium dodecyl sulfate polyacrylamide electrophoresis. Two-dimensional electrophoresis of the pure enzyme resolves three spots (each with molecular weights of 60,000) with isoelectric points of 5.4, 5.8 and 5.9. This charge heterogeneity cannot be explained by the presence of sugar or lipid moieties on the enzyme. Amino acid analysis indicates a relatively high content of hydrophobic amino acids and a lower serine content than other preparations of tyrosine hydroxylase. The enzyme hydroxylates tryptophan at approximately 1% of its rate of tyrosine hydroxylation but will not catalyze the hydroxylation of phenylalanine. Polyclonal antibodies were produced in rabbits against pure tyrosine hydroxylase and were judged to be monospecific by Western blot analysis. The IgG fraction was isolated from serum, and when coupled to cyanogen bromide activated Sepharose, could be used to purify tyrosine hydroxylase from crude extracts in a single step. The antiserum proved to be very useful in immunoprecipitation and immunocytochemical experiments with tyrosine hydroxylase.

Original languageEnglish (US)
Pages (from-to)463-475
Number of pages13
JournalNeurochemistry International
Volume11
Issue number4
DOIs
StatePublished - 1987

All Science Journal Classification (ASJC) codes

  • Cellular and Molecular Neuroscience
  • Cell Biology

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