W.Barry Van Winkle

The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Effects of cyclic AMP-dependent protein kinase and phosphorylase b kinase.

Calcium transport into sarcoplasmic reticulum fragments isolated from dog cardiac and mixed skeletal muscle (quadriceps) and from mixed fast (tibialis), pure fast (caudofemoralis) and pure slow (soleus) skeletal muscles from the cat was studied. Cyclic AMP-dependent protein kinase and phosphorylase b kinase stimulated the rate of calcium transport although some variability was observed. A specific protein kinase inhibitor prevented the effect of protein kinase but not of phosphorylase b kinase. The addition of cyclic AMP to the sarcoplasmic reticulum preparations in the absence of protein kinase had only a slight stimulatory effect despite the presence of endogenous protein kinase. Cyclic AMP-dependent protein kinase catalyzed the phosphorylation of several components present in the sarcoplasmic reticulum fragments; a 19000 to 21 000 dalton peak was phosphorylated with high specific activity in sarcoplasmic reticulum preparations isolated from heart and from slow skeletal muscle, but not from fast skeletal muscle. Phosphorylase b kinase phosphorylated a peak of molecular weight 95000 in all of the preparations. Cyclic AMP-dependent protein kinase-stimulated phosphorylation was optimum at pH 6.8; phosphorylase b kinase phosphorylation had a biphasic curve in cardiac and slow skeletal muscle with optima at pH 6.8 and 8.0. The addition of exogenous phosphorylase b kinase or protein kinase increased the endogenous level of phosphorylation 25-100%. All sarcoplasmic reticulum preparations contained varying amounts of adenylate cyclase, phosphorylase b and a (b:a = 30.1), debrancher enzyme and glycogen (0.3 mg/mg protein), as well as varying amounts of protein kinase and phosphorylase b kinase which were responsible for a significant endogenous phosphorylation. Thus, the two phosphorylating enzymes stimulated calcium uptake in the sarcoplasmic reticulum of a variety of muscles possessing different physiologic characteristics and different responses to drugs. In addition, the phosphorylation catalyzed by these enzymes occurred at two different protein moieties which make physiologic interpretation of the role of phosphorylation difficult. While the role phosphorylation in these mechanisms is complex, the presence of a glycogenolytic enzyme system may be an important link in this phenomenon. The sarcoplasmic reticulum represents a new substrate for phosphorylase b kinase.

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: A possible role in myocardial ischemia

Abstract Palmitylcarnitine is a time-dependent inhibitor of the Ca 2 + -ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.

Association of glycogenolysis with cardiac sarcoplasmic recticulum: II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischemia: Evidence for a phosphorylase kinase membrane complex

The glycogenolytic enzymes associated with cardiac sarcoplasmic reticulum have been shown to have two major features which make the munique. The conversion of phosphorylase b to phosphorylase a can take place in the presence of 20 m m eGTA which ordinarily inhibits this reaction by chelating the calcium which is required for the enzyme phosphorylase b kinase of muscle. In addition, inhibitors of “debrancher” enzyme such as Tris also inhibit phosphorylase b activity in the sarcoplasmic reticulum-glycogen complex, but do not inhibit purified phosphorylase. The phosphorylase b to a conversion which occurs through the addition of ATP alone is very rapid, being complete within 5–10 min in normal cardiac sarcoplasmic reticulum and converting approximately 30% of the total phosphorylase activity. DOC solubilization irreversibly perturbed the sarcoplasmic reticulum membrane so that EGTA resistance could not be reconstituted although phosphorylase b to a conversion continued to some extent. Thus, EGTA sensitivity depended, at least in part, on some specific property of the native membrane system. Examination of skeletal muscle sarcoplasmic reticulum from fast, slow and mixed skeletal muscle revealed that phosphorylase b to a conversion occurred, but was EGTA sensitive in a manner similar to that of the solubilized cardiac preparation. Glycogen depletion solubilized phosphorylase and uncoupled it from its activating enzymes. This uncoupling could not be reversed by the addition of glycogen at any concentration. A relationship was demonstrated between the phosphorylase activity in sarcoplasmic reticulum fractions and the glycogen concentration in both normal sarcoplasmic reticulum that was amylase treated, and from sarcoplasmic reticulum which was isolated from ischemic tissue. A correlation between density observed on a sucrose density gradient and glycogen concentration was described. The relationship between phosphorylase and debrancher activity depended only on the presence of glycogen and therefore was not as specific a structural feature. The results suggest that the sarcoplasmic reticulum glycogen complex is highly specific and that specific functional couplings between glycogen and sarcoplasmic reticulum membranes exist. Thus, modulation of the complex could occur through control of glycogenolysis or alterations in membrane conformation, and this complex might represent a link between energy metabolism and excitation-contraction coupling.

Action of lecithin:Cholesterol acyltransferase on model lipoproteins: Preparation and characterization of model nascent high density lipoprotein

Apolipoprotein A-I, the major protein of human plasma high density lipoprotein, is the primary activator of plasma lecithin:cholesterol acyltransferase. In vitro, the association of apolipoprotein A-I with physiological phosphatidylcholines can be catalyzed by mixing the protein and lipid with sodium cholate, which is removed by chromatography. The apolipoprotein A-I/phospholipid complex has the physical properties of an HDL, and when cholesterol is present the complex is a highly reactive substrate in the lecithin:cholesterol acyltransferase-catalyzed reaction. The relative reactivity of this complex compared with a number of other lipid-protein complexes is presented and discussed.

The nature of the transport ATPase—Digitalis complex: III. Rapid binding studies and effects of ligands on the formation and stability of magnesium plus phosphate-induced glycoside—Enzyme complex

Abstract The [ 3 H]ouabain-Na + ,K + -ATPase complex formed in the presence of magnesium and inorganic phosphate (Complex II) may represent a different conformation than that formed in the presence of ATP, magnesium, and sodium (Complex I). The nomenclature, Complex I and II, are operational in nature. The differences in binding may, e.g., be due to different levels of phosphorylation and/or to different conformations of the enzyme prior to binding. The kinetics of binding under conditions that lead to Complex II formation can be characterized by a hyperbolic curve arbitrarily divided into three phases. Sodium and potassium exert differential effects on these three phases of binding, with sodium having a greater inhibitory effect than potassium. Potassium primarily retards the reaction. When added to the binding medium (Complex II) after maximal binding, sodium and potassium are found not only to dissociate a portion of the glycoside-enzyme complex, but also appear to convert the unbound enzyme into a conformation which may bind only a small amount of glycoside.

Unsaturated aminophospholipids are preferentially retained by the fast skeletal muscle CaATPase during detergent solubilization. Evidence for a specific association between aminophospholipids and the calcium pump protein

When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.

Cyclic AMP modulation of calcium accumulation by sarcoplasmic reticulum from fast skeletal muscle.

The role of cyclic 3,5-AMP in modulating sarcoplasmic reticulum from fast skeletal muscle was studied. The rate of Ca2+ uptake was stimulated in the presence of protein kinase plus 1 micron cyclic AMP. The stimulation was absent when denatured protein kinase was used. When an adenylate cyclase inhibitor was added, the uptake rates fell to 55% of control. This decrease in rate was partially overcome by 1 micron cyclic AMP. A modulating role for cyclic AMP in fast skeletal muscle is proposed.

Cytochemical studies of a glycogen-sarcoplasmic reticulum complex

SummaryEnzymatically active cardiac sarcoplasmic reticulum (SR) fractions contain glycogen. Previous biochemical and morphological studies indicate that the glycogen particles are membrane associated. In the present study, further evidence for membrane-associated glycogen particles in these cardiac SR fractions is presented: (1) morphological parameters, (2) enzymatic digestion by glucoamylase and alpha-amylase and (3) cytochemical staining by two different methods. Dense granules comparable in size (20–30 nm diameter), electron density and substructure to glycogen particles observed in intact cardiac muscle and in glycogen preparations isolated from skeletal muscle were seen. Most of these glycogen particles were removed by amylase digestion except for glycogen particles closely adhering to vesicle membranes. Two different cytochemical techniques (bismuth subnitrate and silver proteinate) revealed a positive reaction product over the glycogen particles. These findings provide further support for the biochemical finding of a structured enzyme complex involving the SR, glycogenolytic enzymes and glycogen.

On the lack of inotropy of cardiac glycosides on skeletal muscle: A comparison of Na+,K+-ATPases from skeletal and cardiac muscle☆

Abstract Comparison of Na,K-ATPase from skeletal and cardiac muscle revealed that, although the skeletal muscle enzyme was only slightly less sensitive to inhibition by ouabain, the rates of [ 3 H]ouabain binding to, and dissociation from, the skeletal enzyme were much faster than the corresponding rates for the cardiac enzyme. The skeletal muscle enzyme required higher concentrations of potassium to stabilize the ouabainenzyme complex and to stimulate the K + -phosphatase activity. The K + -phosphatase activity was only 8% of the Na,K-ATPase activity of the skeletal muscle enzyme, compared to 22% for the cardiac preparation. The glycoprotein subunit found in Na,K-ATPases from cardiac and many other tissues appeared to be absent in the enzyme from skeletal muscle. The differences in binding and dissociation rates for ouabain suggest that there may be significant differences in the structure of the digitalis receptor in the two enzymes. The I 50 for ouabain inhibition of the skeletal muscle Na,K-ATPase was, however, only slightly higher than for the cardiac enzyme, suggesting that the lack of an inotropic effect of cardiac glycosides on skeletal muscle could not be due to failure of the digitalis drugs to bind to and inhibit the membrane-linked sodium pump.

Time-dependent resistance to alkaline pH of oxalate-supported calcium uptake by sarcoplasmic reticulum

Abstract Both oxalate-supported Ca 2+ uptake and Ca 2+ -stimulated ATPase activity of the sarcoplasmic reticulum are sensitive to the pH of the assay medium. Ca 2+ uptake is optimal at relatively acidic pH (6.2–6.6); whereas, Ca 2+ -stimulated ATPase activity is optimal at a more alkaline pH (7.4–8.0). Following the addition of ATP, Ca 2+ uptake demonstrates a time-dependent resistance to the inhibition by an alkaline pH. Once the linear phase of Ca 2+ uptake is reached, alkalinization thereafter does not alter the rate established at the acidic pH. A similar time-dependent resistance is observed to the inhibition of Ca 2+ uptake by the cation ionophore, X537A. In contrast, acidification of the alkaline medium after Ca 2+ uptake is initiated by ATP has no such resistance to change. Acidification results in a prompt acceleration of the rate of Ca 2+ uptake identical to that observed under control conditions at the acidic pH. Ca 2+ -stimulated ATPase activity, however, increases with alkalinization and decreased with acidification, regardless of time, in a manner expected from the rates observed under conditions when the pH is constant from the time of ATP addition. The results suggest that there is a time-dependent, pH-sensitive factor of oxalate-supported Ca 2+ uptake. This factor can be activated by acidification at any time after ATP addition and, thus, does not represent a destruction of membrane function. In contrast, Ca 2+ -stimulated ATPase activity demonstrates no time-dependent resistance to pH change.