Association of Glyceraldehyde‐3‐Phosphate Dehydrogenase with the Particulate Fraction of Chicken Skeletal Muscle

Abstract

When chicken breast muscle was homogenized in water, approximately 86 % of the glyceraldehyde-3-phosphate dehydrogenase was associated with the particulate fraction.The enzyme was solubilized by increasing pH with a very marked increase in the pH range of 6.9 to 7.1. At low ionic strength (about 0.015), approximately 50 % of the enzyme is solubilized at pH 7.5 and above. Increasing ionic strength also led to increased solubilization. In addition, there was a specific cation effect with Ca2+ > M 8 + > K + > Na+ at a constant ionic strength. Glyceraldehyde 3-phosphate and 2,3-bisphosphoglycerate were effective in partially solubilizing the enzyme. Solubilized glyceraldehyde-3-phosphate dehydrogenase can rebind to the particulate fraction of the homogenized muscle. The soluble form of the enzyme has a higher V and a higher K, (glyceraldehyde 3-phosphate) than the enzyme bound to the particulate fraction.It has been shown that a large number of enzymes can bind to structural parts of the cell. Particulatebound enzymes were detected in plants [l], in microorganisms [2,3], in erythrocytes [4], in brain tissue [5, 61 and in skeletal muscle [7]. Several of the glycolytic enzymes were shown to bind to the glycogen of the cell [8] or to its structural proteins [9,10]. Glyceraldehyde-3-phosphate dehydrogenase, which was shown to be an important control point in glycolysis [ll, 121, was reported to be localized in the sarcoplasmic reticulum [13] and in the isotropic zones of skeletal muscle cells [14]. Arnold and Pette [15] showed that glyceraldehyde-3-phosphate dehydrogenase binds to F-actin in a reversible manner; they also found that such interaction was influenced by changes in pH, ionic strength, and certain cell metabolites. Shin and Carraway [16] showed that glyceraldehyde-3-phosphate dehydrogenase was associated with human erythrocyte membrane and could be released by treatment with detergents, trypsin, and ATP.In the present report, we examine the particulate nature of glyceraldehyde-3-phosphate dehydrogenase in chicken breast muscle and describe the factors that influence its solubilization. Some kinetic parameters of the bound and soluble form of the enzyme are also discussed. MATERIALS AND METHODS Materials~~-Glyceraldehyde-3-phosphate (barium salt), yeast phosphoglycerate kinase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase were purchased from Sigma Chemical Company. AMP, ADP, ATP, NAD' and NADH were purchased from P-L Biochemicals. Cleland's reagent (dithiothreitol), 3-phosphoglyceric acid and reduced glutathione were purchased from Calbiochem. Hens, which served as the source of the skeletal muscle, were obtained from the Department of Veterinary and Animal Sciences at the University of Massachusetts. Birds of mixed breeds and different ages were used. Preparation of Muscle HomogenatesHens were sacrificed by decapitation and the white breast muscle, Pectoralis major, was excised and placed in cold distilled water. The muscle was cut into small cubes and excess fat and connective tissue remov...

Linear relationships between mitochondrial forces and cytoplasmic flows argue for the organized energy‐coupled nature of cellular metabolism

“…These proposals are compatible with the suggested roles of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase as mediators of communication between mitochondrial and cytoplasmic compartments [19,40] provided that these enzymes are membrane-associated [28,29]. Furthermore, they accommodate well the numerous observations of apparent nearequilibrium between cytoplasmic and mitochondrial redox and phosphorylation states [19,41].…”

“…Although the exact form of this organization is uncertain, it is now well-established that many enzymes of intermediary metabolism are membrane-associated [27][28][29][30][31][32] and that enzymes in pathways such as glycolysis can bind reversibly to cytoplasmic substructures with concomitant modulation of their kinetic properties [27,28,[33][34][35][36][37]. It is feasible that enzymes bound in this manner could come within the domain of electric fields generated as a consequence of zip [38].…”

Lysophosphatidylcholine Stimulates the Release of Arachidonic Acid in Human Endothelial Cells

“…This phenomenon was also observed with other enzymes upon their association with membranes and was attributed to a reduced accessibility of exogenous radioactive substrate to the enzyme and/or to a "dilution" effect on the exogenous radioactive substrate; i.e. the presence of endogenous membrane phospholipids lowered the effective specific activity of the radioactive substrate (57)(58)(59). The normal Ca 2ϩ concentration in endothelial cells is approximately 70 nM (60,61), and in the current study lyso-PC was found to cause a 3-fold increase in the intracellular Ca 2ϩ level.…”

Phosphorylation of myofibrillar proteins in post‐mortem ovine muscle with different tenderness

“…Glycolytic enzymes, especially aldolase, glyceraldehyde‐3‐phosphate dehydrogenase and phosphofructokinase, have significant affinity for the myofibrillar apparatus, e.g. actin, troponin and tropomyosin …”