Specificity of the metal-salt method for the localization of Ca2(+)-ATPase activity studied in rat adenohypophysis tissue in a model system of polyacrylamide gel films.
The histochemical method using the lead-salt technique for the detection of Ca2+)-ATPase activity has been studied quantitatively in rat adenohypophyseal cell homogenates incorporated in polyacrylamide gel films using cytochemical conditions which were applied for the ultrastructural localization of this enzyme (El-sherif and Bácsy, 1988, 1989). Polyacrylamide gel films including cell homogenates were fixed in the presence or absence of calcium chloride, incubated for Ca2(+)-ATPase activity, and the mean integrated absorbance was determined. Omission of Ca2+ or levamisole from the incubation medium, as well as substitution of ATP by beta-glycerophosphate resulted in significantly decreased activity. Incubations with media containing AMP or ADP instead of ATP resulted in absorbance values not significantly different from background values obtained with substrate-free media. Maximum absorbance was obtained when 1% CaCl2 was added to the fixative. Absorbance values increased with incubation time up to 45 min. Most of these data correlated well with our previous findings for the ultrastructural localization of Ca2(+)-ATPase activity in rat adenohypophysis and it can be concluded that the method is valid and specific.
el-Sherif
G
G
Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest.
Van Noorden
C J
CJ
Bácsy
E
E
D000180:Acrylic Resins; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D005994:Glycerophosphates / Q000494:pharmacology; D006651:Histocytochemistry; D008297:Male; D008567:Membranes, Artificial; D010903:Pituitary Gland, Anterior / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains
D000180:Acrylic Resins; D005994:Glycerophosphates; D008567:Membranes, Artificial; C016680:polyacrylamide gels; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D002122:Calcium Chloride; D002118:Calcium