Ca2+ Uptake Coupled to Glycogen Phosphorolysis in the Glycogenolytic-Sarcoplasmic Reticulum Complex from Rat Skeletal Muscle M. Nogues, A. Cuenda, F. Henao and C. Gutierrez-Merino Departamento de Bioqufmica y Biologfa Molecular, Facultad de Ciencias, Universidad de Extrem adura, 06080-Badajoz, Spain Z. Naturforsch. 51c, 591-598 (1996); received January 9/February 28, 1996 Ca2+-Uptake, Glycogenolysis, Sarcoplasmic Reticulum, Rat, Skeletal Muscle The glycogenolytic-sarcoplasmic reticulum complex from rat skeletal muscle accumulates Ca2+ upon stimulation of glycogen phosphorolysis in the absence of added ATP. It is shown that an efficient Ca2+ uptake involves the sequential action of glycogen phosphorylase, phos- phoglucomutase and hexokinase, which generate low concentrations of ATP (approximately 1-2 ^m) compartmentalized in the immediate vicinity of the sarcoplasmic reticulum Ca2+, Mg2+-ATPase (the Ca2+ pump). The Ca2+ uptake supported by glycogenolysis in this subcel- lular structure is strongly stimulated by micromolar concentrations of AMP, showing that the glycogen phosphorylase associated with this complex is in the dephosphorylatedb form. The results point out that the flux through this compartmentalized metabolic pathway should be enhanced in physiological conditions leading to increased AMP concentrations in the sarcoplasm, such as long-lasting contractions and in ischemic muscle. Introduction rectly involved in energy metabolism in muscle, Glycogen particles are intimatelly associated such as myokinase, creatine kinase and AMP de­ with sarcoplasmic reticulum (SR) 1 in rabbit skele­ aminase (Cuenda et al. 1994). Early studies with tal muscle (Wanson and Drochmans, 1972, GL-SR have shown that this association allows to Entman et al., 1980). Subcellular fragmentation al­ trigger a faster glycogen phosphorolysis at the on­ lows for preparation of glycogenolytic sarcoplas­ set of skeletal muscle contraction (Entman et al., mic reticulum complexes (Entman et al., 1980). 1980). Moreover, SR membrane preparations from Since the phosphorylated phosphorylase (a rabbit skeletal muscle contain large amounts of form) has low affinity for binding to SR mem­ phosphorylase in its dephosphorylated form (b branes (Cuenda et al., 1991), GL-SR acts as a form), and also glycogen and several enzymes di- phosphorylase b reservoir in skeletal muscle cells due to the large SR membrane network of these cells (Cuenda et al., 1995). The activity of phos­ phorylase b is dependent of AMP and, under physiological conditions which raise the AMP Abbreviations: K 0 5(AMP), concentration of AMP that levels in skeletal muscle sarcoplasm such as anoxia produced 50% of maximum activation; Ap3A , P'P^-di- and during contraction (Busby and Radda, 1976, (adenosine-5') pentaphosphate; Ca2+,Mg2+-ATPase, Ca2+ and Mg2+ dependent adenosine triphosphatase Newsholme and Leech, 1983) glycogen phospho­ (EC 3.6.1.38); EGTA, ethylene glycol bis(ß-aminoethyl rolysis in the vicinity of SR membranes should ether)-N,N'-tetraacetic acid; GL-SR, glycogenolytic- be stimulated. sarcoplasmic reticulum complex; hexokinase, ATP: Using purified SR membranes and purified gly­ D-hexose6 -phosphotransferase (EC 2.7.1.1); IU, amount of enzyme that released1 umol product per min at satu­ cogen phosphorylase b from rabbit skeletal muscle rating substrate concentration; PGM, phosphoglucomu- we have shown that this association can form in tase (a-D-glucose-1,6 phosphomutase, EC 5.4.2.2); phos­ vitro a metabolic shuttle by coupling glycogenoly­ phorylase, glycogen phosphorylase (EC 2.4.1.1); SR, sarcoplasmic reticulum; Tes, 2-[(2-hydroxy-l,l bis-(hy- sis to support Ca2+ uptake through production of droxymethyl) ethylamine]; Tris, tris (hydroxymethyl) ATP in the vicinity of the SR Ca2+,Mg2+-ATPase aminomethane. (Cuenda et al., 1993). This metabolic pathway can Reprint requests to Dr. F. Henao. be written as follows. Fax: 3424271304. 0939-5075/96/0700-0591 $ 06.00 © 1996 Verlag der Zeitschrift für Naturforschung. All rights reserved. D 592 M. Nogues et al. ■ Ca2+-Uptake Coupled to Glycogen Phosphorolysis (Glycogen),,., method (Dubois et al., 1956), using glycogen as standard, and was found to be on average 25 ±5 (Glycogen),, + Pi ► Glc 1 -P ----- (.ig glycogen per mg of protein (n > 10). 1 2 Ca2+ uptake by GL-SR membranes was mea­ sured with the use of 45Ca2+ and Millipore HA ADP Glucose filters as indicated elsewhere (Cuenda et al., 1993). All the data reported in this paper are the average Glc 6-P — ^ — *■ A T P --------- > Ca2+ uptake 3 4 of triplicate experiments carried out with, at least, two different GL-SR preparations. where Glc 1-P and Glc 6-P are glucose-l-phos- phate and glucose-6-phosphate, respectively; and Enzym e assays 1-4 are the enzymes phosphorylase, PGM, hexo- kinase and Ca2+,Mg2+-ATPase, respectively. As The ATPase activity was measured using the noted in (Cuenda et al., 1993), a major bioener- coupled enzyme system pyruvate kinase/lactate getic advantage of this compartmentalized meta­ dehydrogenase as indicated in Cuenda et al. bolic pathway is the efficient use in the cell of low (1990), with the following assay medium; 100 mM concentrations of high energy phosphate in the Tes (pH 7.0)/ 100 mM KC1/ 20% v/v glycerol/ 5 form of ATP generated from low energy phos­ mM MgCl2/ 2.5 mM ATP/ 0.1 mM CaCl2/ 0.25 mM phate compounds, such as Glc 6-P. The flux NADH/ 0.42 mM phosphoe/io/pyruvate/ 7.5 IU through this metabolic pathway will be blocked pyruvate kinase/ 18 IU lactate dehydrogenase/ 5 upon activation of the glycogen phosphorylase b m M NaN3/ 0.4 [Ag/ml calcimycin and 10 [Ag G L-SR kinase at the onset of muscle contraction, because protein ml-1. The Ca2+-dependent ATPase activ­ the phosphorylated a form of glycogen phospho­ ity of different preparations (/7> 10) of the rat rylase has low affinity for binding to SR mem­ GL-SR complex at 25 °C ranged from 0.85 to 1.45 branes (Cuenda et al., 1991). [.imol ATP hydrolyzed per min per mg protein. In this communication we report experimental Unless stated otherwise, the phosphorylase ac­ data showing that our previous results in rabbit tivity had been measured in the direction of glyco­ skeletal muscle can be extended to rat skeletal gen degradation as indicated in Cuenda et al. muscle where a GL-SR subcellular structure is (1991), with the following assay medium: 50 mM also present. Using the isolated GL-SR from rat imidazole (pH 6.9)/ 10 mM magnesium acetate/ 12 skeletal muscle we show that this metabolic shuttle mM KH 2P 0 4/ 0.63 m M NADP+/ 0.45 g-l_1 glyco­ should operate more efficiently in physiological gen/ 50 [am glucose-1,6-diphosphate/ 5 IU PGM/ 5 conditions where the AMP concentration raises in IU glucose-6-phosphate dehydrogenase and 100 the micromolar range, such as ischemic muscle and [Ag GL-SR protein-ml“1. The phosphorylase activ­ in tetanic or long lasting contractions. ity in the presence of 1 mM AMP of different prep­ arations of the rat GL-SR (n ■>10) at 30 °C ranged from 0.12 to 0.72 [imol glucose-l-phosphate pro­ duced per min per mg protein. Materials and Methods The PGM and hexokinase activities were mea­ GL-SR has been prepared from skeletal muscle sured at 35 °C using the coupled enzyme glucose of male Fischer rats, following the protocol of 6-phosphate dehydrogenase with the following as­ Entman et al. (1980). Phosphorylase of rat skeletal say mixture: 100 mM Tes-Tris (pH 7.0)/ 50 mM KC1/ muscle was prepared as described by Gutierrez- 10 mM MgCl2/ 20 mM H2P 0 4K/ 50 [am CaCl2/ 0.48 Merino et al. (1980). The protein concentration mM NADP+/ 1 IU glucose 6-phosphate dehydroge­ was determined using the method of Lowry nase, and 10 mM glucose 1-phosphate/ 50 [im glu­ (Lowry et al., 1951), with bovine serum albumin cose 1.6-diphosphate for PGM activity measure­ as standard. ments or 3 mM glucose/ 2 mM ATP for the The glycogen content of GL-SR preparations hexokinase activity assay medium. Variations in has been measured following the phenol sulfuric NADP+ absorbance were recorded at 340 nm. M. Nogues et al. • Ca_+-Uptake Coupled to Glycogen Phosphorolysis 593 The AMP content of ADP solutions had been Results and Discussion determined using the coupled enzyme AMP-de- Our GL-SR preparations from Fischer rats skel­ aminase as indicated in Tovmasian et al. (1990), etal muscle had a phosphorylase activity which is with a value for the differential extinction coeffi­ dependent upon the presence of AMP and it is (GA m p G IMP) 8 2 03 cient ~ at 265 nm of . x l M ~‘ inhibited by caffeine (Fig. 1A), therefore, indicat­ cm -1. ing that the phosphorylase bound to these com­ plexes is mainly in its b (dephosphorylated) form. This is a property shared with the GL-SR complex Chemicals from rabbit skeletal muscle (Entman et al., 1980). Bovine serum albumin, ADP, AMP, ATP, phos- The GL-SR and the SR from rabbit skeletal mus­ phoenolpyruvate, EGTA, glucose-1,6-diphos­ cle also had myokinase activity (Entman et al., phate, glucose 6-phosphate, Tris (TRIZMA base) 1980; Cuenda et al., 1994), which produces ATP and TES were obtained from Sigma. Glycogen, from AMP and ADP, and this would interfere with Ap5A, NADH, NADP+, calcimycin, pyruvate ki­ the assays of Ca2+ uptake supported by glycogen nase, lactate dehydrogenase, PGM, hexokinase phosphorolysis in the absence of added ATP. We, and glucose 6-phosphate dehydrogenase were thus, confirmed the presence of myokinase activity purchased from Boehringer Mannheim. Hexoki­ of the GL-SR complex from rat skeletal muscle, nase and PGM were sulfate-free by centrifugation. which on average was found to be 0.14 ± 0.05 [imol All the other chemicals used in this study were ADP per min per mg GL-SR protein.