Studies on hormonal induction of alkaline phosphatase in HeLa cell cultures. Kinetic, thermodynamic and electrophoretic properties of induced and base-level enzymes

Nimai K. Ghosh, Adriana Rukenstein, Robert Baltimore, Rody P. Cox

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

As previously described the "induction" of a 5- to 20-fold increase in alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity by hydrocortisone in HeLa 65 cell cultures requires both RNA and protein synthesis; however, immunological methods clearly show that the amount of enzyme protein is not increased. The present study compares the catalytic properties of the base-level and induced alkaline phosphatases. Km values for these enzymes are similar, however, the maximal velocity (V) of induced alkaline phosphatase (27 nmoles/min) is considerably higher than that of base level (1.3 nmoles/min). The first-order rate constant (k3) for decomposition of enzyme substrate (ES) complex to products, of induced alkaline phosphatase is 7- to 17-fold higher than that of base-level alkaline phosphatase. Enzyme kinetics are similar with both phenylphosphate and p-nitrophenyl-phosphate as substrates. Hormone-induced alkaline phosphatase in HeLa 65 cells possesses higher catalytic activity presumably because it can bind with phosphomonoesters to produce a more rapid decomposition of the ES complex. HeLa cell alkaline phosphatase is similar to the placental enzyme with respect to inhibition by L-tryptophan and L-phenylalanine. Inhibition of both base-level and induced HeLa alkaline phosphatase by these L-amino acids is stereospecific, uncompetitive and homosteric. The inhibitor constants (Ki) for these L-amino acids increases with increasing temperature. Values for change of enthalpy (ΔH), free energy (ΔF), and entropy (ΔS) for binding of L-tryptophan to base-level and induced HeLa alkaline phosphatase are similar and both enzymes resemble the placental alkaline phosphatase. Alkaline phosphatase prepared from HeLa 65 cells grown in the presence and absence of hydrocortisone exhibits three isozymes as detected by electrophoretic mobility on starch gel. The isozymes are molecular weight variants that can be separated by Sephadex G-200 gel filtration. A HeLa 71 cell with high constitutive levels of alkaline phosphatase possesses five isozymes on electrophoresis. Multiple molecular forms of base-level and induced alkaline phosphatase are similar and closely resemble those of human placental enzyme.

Original languageEnglish (US)
Pages (from-to)175-185
Number of pages11
JournalBBA - General Subjects
Volume286
Issue number1
DOIs
StatePublished - Nov 24 1972

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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