Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity.

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)

Equilibria and kinetics of ligand binding to the human erythrocyte glucose transporter. Evidence for an alternating conformation model for transport.

Cytochalasin B (CB), n-propyl beta-D-glucopyranoside (PG), and 4,6-O-ethylidene-D-glucose (EG) are known to bind asymmetrically to the human erythrocyte glucose transporter. The first two compounds bind to the inner (cytoplasmic) surface of the transporter, while the latter binds to the outer surface. Equilibrium measurements of the inhibition of CB binding to the glucose transporter reported herein indicate that the ternary complexes of CB transporter with EG, PG, or D-glucose are not formed. Moreover, measurements of CB binding in the presence of both EG and PG or in the presence of high concentrations of D-glucose show that a ternary complex of transporter and sugars bound simultaneously on both sides of the membrane probably does not occur. Finally, the kinetics of dissociation of radiolabeled CB from the transporter in the presence of CB, glucose, PG, and EG have been determined. With the exception of the case of EG, the kinetics fit a simple scheme of rate-limiting unimolecular dissociation, and in no instance do they suggest the existence of a ternary complex of sugar, CB, and transporter. These data are consistent with a model for transport in which the substrate binding site exists alternately at the cytoplasmic and external faces of the membrane, as the result of protein conformational change.

Changes in the intrinsic fluorescence of the human erythrocyte monosaccharide transporter upon ligand binding.

The effect of ligands on the tryptophan fluorescence of the purified monosaccharide transporter from human erythrocytes has been investigated. Cytochalasin B, D-glucose, and ethylideneglucose quench the fluorescence of the protein at longer wavelengths by 17%, 13%, and 8%, respectively. Propyl glucoside, another ligand, has no effect on the protein fluorescence. Values of the dissociation constants for cytochalasin B, D-glucose, and ethylideneglucose were determined from the concentration dependence of fluorescence change; these agree with the values obtained from the effects of these compounds upon the binding of [3H]cytochalasin B measured by equilibrium dialysis. There is no correlation between the effect of each ligand on the fluorescence of the transporter and the conformational state expected for its complex on the basis of other evidence. The fact that the quenching is greatest at longer wavelengths suggests that an exposed tryptophan residue(s), possible located at the ligand binding sites, is the perturbed one.

The elevation of cellular phosphatidic acid levels caused by polyomavirus transformation can be disassociated from the activation of phospholipase D.

Middle T (mT), the oncogene of murine polyomavirus, causes transformation of rat fibroblasts by activating a number of signal transducing pathways usually used by polypeptide growth factors and their receptors. Here, we report data regarding the activation of signal transducing pathways involving phospholipase D (PL-D). The hydrolysis of phospholipids by PL-D produces phosphatidic acid (PA), a compound with multiple biological effects. The PA content of cells expressing wild-type mT, introduced via a number of different methods, is approximately 50% higher than their untransformed counterparts. This increase in cellular PA content is associated with an approximately 65% increase in PL-D activity in cells expressing wild-type mT. We have also examined the effects of a number of site-directed mutants of mT, on both cellular PA levels and on PL-D activity. Mutants that do not produce mT (Py808A) or that produce a truncated, nonmembrane bound mT (Py1387T) have PA levels similar to that of control cells. Cells expressing the 322YF mutant of mT (which abolishes interaction of mT with phospholipase C gamma1) show increases in both PA levels and PL-D activity that are similar to those seen with wild-type mT. Expression of mutants that abolish the interaction of mT with either shc or with phosphatidylinositol 3-kinase (250YS and 315YF, respectively) cause an increase in PL-D activity comparable to that seen with wild-type mT. However, the PA content of cells expressing these mutants is not elevated. These results suggest that mT causes activation of cellular PL-D, but this activation alone is not sufficient to cause an increase in cellular PA content. Therefore, wild-type mT must affect another, as yet unknown, step in PA metabolism.

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.

Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm.

Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances.

Phosphatidylinositol 3-phosphate is present in normal and transformed fibroblasts and is resistant to hydrolysis by bovine brain phospholipase C II.

The transforming protein of polyoma virus, middle T antigen, associates with two cellular enzymes, pp60c-src, a protein tyrosine kinase, and a phosphatidylinositol kinase that forms phosphatidylinositol 3-phosphate. The formation of a ternary complex of these proteins is essential for complete transformation and maximal tumor induction by the virus. A mutant virus encoding an altered middle T protein that activates pp60c-src but fails to bind phosphatidylinositol kinase is partially defective in transformation. We have confirmed, using an enzymological method, that the product of the in vitro reaction catalyzed by middle T-pp60c-src-phosphatidylinositol kinase complexes is phosphatidylinositol 3-phosphate (PtdIns(3)P), as previously reported (Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 332, 644-646). PtdIns(3)P is present in normal as well as virus-infected and transformed cells at levels ranging from 0.6 to 2.6% of the major phosphatidylinositol phosphate isomer, phosphatidylinositol 4-phosphate (PtdIns(4)P). Steady-state levels of PtdIns(3)P do not appear to be affected by the expression of middle T in cells. PtdIns(3)P is not hydrolyzed by bovine brain phospholipase C II, which readily cleaves PtdIns(4)P and other phosphatidylinositols. This result underscores the likelihood that the metabolism of PtdIns(3)P is distinct from that of PtdIns(4)P and raises further questions regarding a possible role of PtdIns(3)P in normal and neoplastic cell growth.