A study of the phosphorylated intermediate of sarcoplasmic reticulum atpase

G. Inesi, E. Maring, A. J. Murphy, Bentson McFarland

Research output: Contribution to journalArticle

87 Citations (Scopus)

Abstract

Incorporation of ATP terminal phosphate into sarcoplasmic reticulum (SR) occurs very rapidly on ice. Ca2+ is a requirement and half-maximal activation is obtained in the presence of 1 × 10-7, m Ca2+. The activating Ca2+ concentrations are identical for phosphate incorporation and ATPase, while excess Ca2+ only inhibits ATPase. The rate of enzymatic degradation of the 32P-membrane complex, in the presence of Mg2+, is comparable to that of ATP hydrolysis. Mg++ accelerates the P- membrane complex turnover. The pH stability of the phosphorylated species is different in membranes denatured at acid pH (trichloroacetic acid), as opposed to membranes denatured at neutral pH (Salyrgan, acetone) and then exposed to various pH values. ATPase activity and steady-state levels of 32P-membrane complex are reduced when SR is incubated in the presence of hydroxylamine. However, SR incubated with hydroxylamine in optimal conditions for formation of P-membrane complex, regains full activity after washings. This lack of permanent inactivation suggests that hydroxylamine does not form a stable hydroxamate at the active site. Degradation of trichloroacetic acid-treated 32P-membrane complex is accelerated by hydroxylamine. On the other hand, 0-(14C) methylhydroxylamine is bound by trichloroacetic acid-treated SR, independent of its previous incubation in the presence or in the absence of ATP. This indicates that the hydroxylamine effect may not be related to a specific reaction with an acylphosphate. Solubilization of 32P-membrane complex with Triton X 100 at acid pH, and gel chromatography, yield phosphorylated particles with 60-80% of the original specific activity. On the other hand, particles solubilized with deoxycholate at neutral pH retain only traces of 32P. A mechanism is proposed for SR ATPase, in which Ca2+ binding to the membrane (a) occurs as a consequence of site modification induced by ATP binding, and (b) activates phosphoryl transfer and formation of a phosphorylated intermediate.

Original languageEnglish (US)
Pages (from-to)285-294
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume138
Issue number1
DOIs
StatePublished - 1970
Externally publishedYes

Fingerprint

Sarcoplasmic Reticulum
Adenosine Triphosphatases
Hydroxylamine
Membranes
Trichloroacetic Acid
Adenosine Triphosphate
Mersalyl
Phosphates
Degradation
Regain
Acids
Deoxycholic Acid
Octoxynol
Ice
Acetone
Chromatography
Washing
Gel Chromatography
Hydrolysis
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

A study of the phosphorylated intermediate of sarcoplasmic reticulum atpase. / Inesi, G.; Maring, E.; Murphy, A. J.; McFarland, Bentson.

In: Archives of Biochemistry and Biophysics, Vol. 138, No. 1, 1970, p. 285-294.

Research output: Contribution to journalArticle

Inesi, G. ; Maring, E. ; Murphy, A. J. ; McFarland, Bentson. / A study of the phosphorylated intermediate of sarcoplasmic reticulum atpase. In: Archives of Biochemistry and Biophysics. 1970 ; Vol. 138, No. 1. pp. 285-294.
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abstract = "Incorporation of ATP terminal phosphate into sarcoplasmic reticulum (SR) occurs very rapidly on ice. Ca2+ is a requirement and half-maximal activation is obtained in the presence of 1 × 10-7, m Ca2+. The activating Ca2+ concentrations are identical for phosphate incorporation and ATPase, while excess Ca2+ only inhibits ATPase. The rate of enzymatic degradation of the 32P-membrane complex, in the presence of Mg2+, is comparable to that of ATP hydrolysis. Mg++ accelerates the P- membrane complex turnover. The pH stability of the phosphorylated species is different in membranes denatured at acid pH (trichloroacetic acid), as opposed to membranes denatured at neutral pH (Salyrgan, acetone) and then exposed to various pH values. ATPase activity and steady-state levels of 32P-membrane complex are reduced when SR is incubated in the presence of hydroxylamine. However, SR incubated with hydroxylamine in optimal conditions for formation of P-membrane complex, regains full activity after washings. This lack of permanent inactivation suggests that hydroxylamine does not form a stable hydroxamate at the active site. Degradation of trichloroacetic acid-treated 32P-membrane complex is accelerated by hydroxylamine. On the other hand, 0-(14C) methylhydroxylamine is bound by trichloroacetic acid-treated SR, independent of its previous incubation in the presence or in the absence of ATP. This indicates that the hydroxylamine effect may not be related to a specific reaction with an acylphosphate. Solubilization of 32P-membrane complex with Triton X 100 at acid pH, and gel chromatography, yield phosphorylated particles with 60-80{\%} of the original specific activity. On the other hand, particles solubilized with deoxycholate at neutral pH retain only traces of 32P. A mechanism is proposed for SR ATPase, in which Ca2+ binding to the membrane (a) occurs as a consequence of site modification induced by ATP binding, and (b) activates phosphoryl transfer and formation of a phosphorylated intermediate.",
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