Regulation of transverse tubule ecto-ATPase activity in chicken skeletal muscle.
Transverse tubule (T-tubule) ecto-ATPase from chicken skeletal muscle is an integral membrane glycoprotein that seems to exist as a homodimer and exhibits unusual properties. Treatment of T-tubule membranes with concanavalin A (Con A) did not significantly affect the thermal variation of the fluorescence anisotropy of vesicles labelled with 1,6-diphenyl-1,3,5-hexatriene or trimethylammonium-1,6-diphenyl-1,3,5-hexatriene. Cross-linking of membrane components with glutaraldehyde elicited effects on ecto-ATPase activity very similar to those of Con A treatment: a severalfold increase in activity, a decrease in Triton X-100 sensitivity and a requirement to be present before ATP to exert its action. In addition, glutaraldehyde and Con A normalized the temperature dependence and the kinetic behaviour of the enzyme. Membrane-perturbing agents (detergents, alcohols and cholesterol oxidase), with the sole exception of digitonin, caused a marked decrease in ecto-ATPase activity; the prior presence of Con A prevented this inhibition, whereas when the lectin was added after the membrane perturbing agent, recovery of the activity was not always possible. The addition of nucleotides before Con A led to a suppression of ecto-ATPase stimulation; it occurred when the nucleotide was hydrolysed (ATP or UTP) and when it was not (adenosine 5'-[beta,gamma-imido]triphosphate) and even in the presence of 3 mM P(i). A model is proposed for the complex regulatory mechanisms of chicken T-tubule ecto-ATPase that involves the occurrence of two different catalytic states in an equilibrium modulated by lectins and cross-linking agents, by the structure of the membrane and by the presence of ligands for a regulatory site.
Megías
A
A
Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University, 28040-Madrid, Spain.
Martínez-Senac
M M
MM
Delgado
J
J
Saborido
A
A
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D003208:Concanavalin A / Q000494:pharmacology; D005976:Glutaral / Q000737:chemistry; D050356:Lipid Metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D013379:Substrate Specificity; D013696:Temperature
D003208:Concanavalin A; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D005976:Glutaral