Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm - Inhibition by adenosine

D. Stengel, D. Henry, S. Tomova, A. Borsodi, J. Hanoune

Research output: Contribution to journalArticle

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Abstract

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by α-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 μmol cyclic AMP formed mg protein-1 min-1 at 30°C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30°C, 20°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4° C. The purified enzyme exhibited a K(m) value similar to that of the native enzyme (K(m) = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10818-10826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.

Original languageEnglish
Pages (from-to)241-247
Number of pages7
JournalEuropean Journal of Biochemistry
Volume161
Issue number1
DOIs
Publication statusPublished - 1986

Fingerprint

Adenylyl Cyclases
Adenosine
Purification
Spermatozoa
Catalytic Domain
Enzymes
Durapatite
Adenosine Triphosphate
Proteins
Chymotrypsin
High performance liquid chromatography
Molecular mass
Manganese
Bovine Serum Albumin
Chromatography
Electrophoresis
Sodium Dodecyl Sulfate
Detergents
Cyclic AMP
Gels

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm - Inhibition by adenosine. / Stengel, D.; Henry, D.; Tomova, S.; Borsodi, A.; Hanoune, J.

In: European Journal of Biochemistry, Vol. 161, No. 1, 1986, p. 241-247.

Research output: Contribution to journalArticle

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abstract = "Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by α-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10{\%} and a final specific activity of 19 μmol cyclic AMP formed mg protein-1 min-1 at 30°C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30°C, 20°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80{\%} of the activity remaining after 24 h at 4° C. The purified enzyme exhibited a K(m) value similar to that of the native enzyme (K(m) = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10818-10826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.",
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AU - Hanoune, J.

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N2 - Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by α-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 μmol cyclic AMP formed mg protein-1 min-1 at 30°C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30°C, 20°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4° C. The purified enzyme exhibited a K(m) value similar to that of the native enzyme (K(m) = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10818-10826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.

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