Molecular and Cellular Biochemistry

, Volume 79, Issue 2, pp 161–169 | Cite as

Identification and characterization of a Mg+2-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis

  • Subir K. NagDas
  • Shyamali Mukherjee
  • Barsanjit Mazumder
  • Parimal C. Sen
Original Article

Abstract

Rat testicular microsomal membrane fraction contains both Mg+2-dependent and Mg+2-independent Ca+2-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg+2-independent Ca+2-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37°C and for latter one it is 40°C. Among all the nuclestides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn+2, Ba+2 and Mn+2 have been found to inhibit Mg+2-dependent Ca+2-ATPase activity whereas Mg+2-independent Ca+2-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadata, EDTA and EGTA. I50, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydral groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.

Key words

Mg+2-dependent and Mg+2-independent Ca+2-ATPase microsomal membranes testis (rat) 

Abbreviations

Tris

Tris (hydroxymethyl) aminoethane

Hepes

N-2-hydroxyethyl piperizine-N′-2-ethane sulphonic acid

Pipes

Piperizine-N, N′-bis (2-ethane sulphonic acid)

EGTA

Ethylene Glycol-bis (β-amino ethyl ether) N, N, N′, N′-Tetraacetic Acid

EDTA

Ethylene diamine tetraacetic acid, sodium salt

NEM

N-ethyl maleimide

DTNB

5,5′-dithiobis-(2-nitrobenzoic acid)

CPZ

chlorpromazine hydrochloride

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Copyright information

© Martinus Nijhoff Publishers 1988

Authors and Affiliations

  • Subir K. NagDas
    • 1
  • Shyamali Mukherjee
    • 1
  • Barsanjit Mazumder
    • 1
  • Parimal C. Sen
    • 1
  1. 1.Department of ChemistryBose InstituteCalcuttaIndia

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