Characteristics of multiple forms of the acidic triacylglycerol lipase(s) of canine cardiac myocytes.
Acidic lipase activity was extracted by digitonin treatment from particulate fractions prepared from isolated adult canine myocytes. Both methylumbelliferyloleate (MUO) and trioleoylglycerol were hydrolyzed with an apparent Km of 13 and 135 microM, respectively. The primary products of trioleoylglycerol lipolysis were oleic acid and 1,2-dioleoylglycerol. Hydrolysis of either MUO or triacylglycerol was stimulated in vitro by the addition of cardiolipin or Triton X-100. Triton X-100 alone was sufficient for maximal stimulation of MUO hydrolysis, but cardiolipin further stimulated triacylglycerol lipolysis in the presence of an optimal concentration of Triton X-100. Cardiolipin increased the Vmax without altering the Km for trioleoylglycerol. Upon gel filtration chromatography the 4-methylumbelliferyloleate and triacylglycerol lipase activities eluted in regions consistent with molecular weights of approx. 47 000 and 55 000, respectively. Chromatofocusing revealed predominantly one form of acidic 4-methylumbelliferyloleate hydrolase (pI approx. 6.3), whereas acidic triacylglycerol lipase activity eluted continuously in the pH gradient from 7.2 to 4.3 with no clearly predominant peak of activity. Two forms of both 4-methylumbelliferyloleate and triacylglycerol lipase were eluted from columns of carboxymethyl Bio-Gel at pH 5.7; one form of each lipase activity was not bound and another form of each lipase was eluted with 50-60 mM KCl. The non-bound forms of each lipase were indistinguishable from their respective carboxymethyl-bound forms on the basis of pH dependency or kinetically (similar Km). The non-bound and carboxymethyl-bound peaks of lipolytic activity differed in the ratios of 4-methylumbelliferyloleate hydrolase to triacylglycerol lipase activity. The results suggest that the cardiac myocyte contains multiple forms of acidic lipase, and that the catalytic units primarily responsible for the hydrolysis of methylumbelliferyl esters and triacylglycerols may not be identical.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000145:classification / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology*; D017830:Octoxynol; D010743:Phospholipids / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology
D010743:Phospholipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D008049:Lipase