Membrane-associated guanylate cyclase in cardiac muscle: Regulation by Mg2+ and Ca2+

42 MEMBRANE-ASSOCIATED GUANYLATE CYCLASE IN CARDIAC MUSCLE: REGULATION BY Mg 2+ AND Ca 2+. N. Narayanan and P.Vo Sulakhe. Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada. Earlier we reported that considerable amounts of guanylate cyclase (GC) are present in cardiac sarcolemma (SL). Further, we suggested that GC is a metallo-protein with Mn 2+ bound to the enzyme (which is essential for its activity) and Mg 2+ supports its catalysis whereas Ca 2+ ions exert regulatory influence. Previous work from various laboratories on GC from cardiac and other tissues has been carried out with Mn 2+ and may be of limited physiological significance. In this study, we show that guinea pig heart microsomal fraction (containing SL) contains Mg2+-supported GC that is stimulated by Ca 2+ (maximal stimulation (50%) at i pM Ca 2+, 0.5 mM Mg-GTp2-), and of higher spe~ific activity (5 to 10-fold g~eater compared to homogenate or other primary subcellu]ar fractions). The Mg 2+supported GC required free (excess) Mg 2+ for maximal activity, had an apparent K m of 0. i mM (Mg-GTp2-), was solubil[zed with Triton X-100 and displayed Michaelis-Menten Kinetics. In the presence of low Mn 2+ (0.i mM), Ca2+' failed to stimulate Mg2+-supported GC but, in fact, caused inhibition of GC (50% inhibition at 0.2 mM Ca2+). Other properties of Mg2+-supported GC such as pll- and temperature-dependence, effects of monovalent ions and cholinergic agonists would be presented. [Supported by a grant from Saskatchewan IIeart Foundation (Sulakhe) and post-doctoral fellowship award from Canadian Heart Foundation (Narayanan)].

INHIBITION OF MYOCARDIAL PROTEIN DEGRADATIONBY TLCK. G.V. O'Gorman and K. Wildenthal. University of Texas Health Science Center at Dallas, Dallas, Texas. In addition to cathepsin D (a carboxyl acid proteinase of lysosomal origin, which is the best characterized myocardial proteinase to date), the heart contains other lysosomal protolytic enzymes (e.g., cathepsin BI) as well ~s non-lysosomal, neutral and alkaline proteinases. The relative importance of these various types of enzymes in mediating myocardial protein degradation is unknown. Accordingly, we tested the effect of l-chloro-3-tosylamido-7-amino-2 heptanone (TLCK), an agent known to inhibit trypsin-like enzymes and some thiol proteinases including cathepsin Bl, without inhibiting carboxyl proteinase activity. Rates of myocardial protein degradation in cultured fetal mouse hearts were measured by 2 techniques (rate of loss of radioactivity from TCAprecipitable protein after pre-labeling with tritiated phenylalanine; and rate of lois of cold phenylalanine during cuitivatio, in Lhe presence of cycloheximide to block synthesis). The rate of protein degradation in control hearts, as estimated by the f i r s t technique, was 44 • 3%/24 hrs. TLCK(lO0 ~M) decreased this rate to 35 • 4%/24 hrs (p