Chromatographic characterization of nitrobenzylthioinosine binding proteins in band 4.5 of human erythrocytes: purification of a 40 kDa truncated nucleoside transporter.

DEAE-column-purified band 4.5 polypeptides of human erythrocyte membranes are mostly glucose transporters with nucleoside transporters as a minor component. The purpose of the present work was to differentially identify and isolate the nucleoside transporters in band 4.5 free from glucose transporters. Equilibrium binding studies demonstrated that the band 4.5 preparation binds nibrobenzylthioinosine (NBTI), a potent nucleoside transport inhibitor, at two distinct sites, one with a high affinity (dissociation constant, KD of 1 nM) with a small capacity, BT (0.4 nmol/mg protein), and the other with a low affinity (KD of 15 microM) with a large BT (14-16 nmol/mg protein). The BT of the low-affinity site was equal to that of the cytochalasin B binding site in the preparation. A gel-filtration chromatography of band 4.5 photolabeled with [3H]NBTI and [3H]cytochalasin B identified three polypeptides of apparent Mr 55,000, 50,000 and 40,000. Of these, the 55 kDa polypeptide was specifically labeled by cytochalasin B (p55GT), indicating that it is a glucose transporter. Both the 50 and 40 kDa polypeptides were labeled with NBTI at low ligand concentrations (less than 0.1 microM), which was abolished by an excess (20 microM) of nitrobenzylthioguanosine, indicating that they are two forms (p50NT and p40NT, respectively) of the high affinity NBTI binding protein or nucleoside transporter. At higher (not less than 10 microM) NBTI concentrations, however, p55GT was also labeled with NBTI, indicating that the low-affinity NBTI binding is due to a glucose transporter. Treatment of band 4.5 with trypsin reduced the p50NT labeling with a concomitant and stoichiometric increase in the p40NT NBTI labeling without affecting the high-affinity NBTI binding of the preparation. These findings indicate that the nucleoside transporter is slightly smaller in mass than the glucose transporter and that trypsin digestion produces a truncated nucleoside transporter of apparent Mr 40,000 which retains the high-affinity NBTI binding activity of intact nucleoside transporter. Both p55GT and p50 NT were coeluted in a major protein fraction, P1 in the chromatography, while p40NT was eluted separately as a minor protein fraction, P1a. All three polypeptides formed mixed dimers, which were eluted in a fraction PO. We have purified and partially characterized the truncated nucleoside transporter, p40NT. The purified p40NT may be useful for biochemical characterization of the nucleoside transporter.

Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.

Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.

Solubilization of a high affinity CA-ATPase from dog antrum smooth muscle.

A Mg-independent high affinity Ca-ATPase has recently been reported to be present in the plasma membranes of smooth muscle. This enzyme has now been solubilized using deoxycholate. The membrane-bound and the solubilized enzymes resemble each other in Km for Ca2+, and inhibition by fluphenazine. The solubilized enzyme is, however, more sensitive to inhibition by Mg2+ than the membrane bound enzyme. Radiation inactivation analysis shows that whereas the membrane-bound enzyme had a target size of 98,000 +/- 4,000 Daltons, the solubilized enzyme was only 70,000, +/- 7,000 Daltons.

Target size of Ca-pumps in pig coronary artery smooth muscle.

The target sizes of the oxalate-independent Ca uptake by the plasma membrane enriched fraction F2, and the oxalate-stimulated Ca uptake by a fraction F3 slightly enriched in the endoplasmic reticulum were determined by radiation inactivation. The oxalate-independent Ca uptake was inactivated with a D37 value of 1.96 +/- 0.30 Mrad but the oxalate-stimulated Ca uptake had a D37 value of 0.45 +/- 0.07 Mrad. Thus, in the smooth muscle the oxalate-stimulated Ca uptake appeared to be due to a structure 3 to 6 times larger than was the oxalate-independent Ca uptake. The subcellular site of the ATP-dependent azide insensitive Ca uptake in the smooth muscle has been disputed in the past. It has been suggested to be plasma membrane (PM) by several workers, and endoplasmic reticulum (ER) by others. Recently, however, there has been substantial evidence to support the hypothesis that one Ca uptake system, unaffected by oxalate, resides in the PM and another, stimulated by oxalate, is located in the ER of the smooth muscle. The evidence has been reviewed recently. Here, we show that the two modes of Ca uptake differ in their target sizes as well. To our knowledge, this is the first report on the use of radiation inactivation to distinguish between the two modes of Ca uptake in any tissue.

Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle.

Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.

Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools.

The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.

Determination of the oligomeric structure of the Ca2+ pump protein in canine cardiac sarcoplasmic reticulum membranes using radiation inactivation analysis.

The size of the Ca2+ pump protein in canine cardiac sarcoplasmic reticulum (SR) membranes was determined using target theory analysis of radiation-inactivation data. Samples of cardiac SR were irradiated in the frozen state with increasing doses of high energy electrons from a Van de Graaff accelerator. The loss of Ca2+-dependent ATPase activity and of ATP-dependent, oxalate-facilitated Ca2+ uptake (loading) with increasing irradiation dosage paralleled one another. Also, the loss of staining intensity of the predominant polypeptide (Mr = 110,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis correlated with the loss of ATPase and loading activities. The target size by all three measurements varied between 213,000 and 229,000 Da. We conclude that the Ca2+ pump protein in canine cardiac SR is a dimer.

An ATP-modulated specific association of glyceraldehyde-3-phosphate dehydrogenase with human erythrocyte glucose transporter.

Glyceraldehyde-3-phosphate dehydrogenase was found to bind in vitro to purified, human erythrocyte glucose transporter reconstituted into vesicles. Mild tryptic digestion of the glucose transporter totally inactivated the binding, suggesting that the cytoplasmic domain of the transporter is involved in the binding to glyceraldehyde-3-phosphate dehydrogenase. The binding was abolished in the presence of antisera raised against the purified glucose transporter, further supporting specificity of this interaction. The binding was reversible with a dissociation constant (Kd) of 3.3 x 10(-6) M and a total capacity (Bt) of approximately 30 nmol/mg of protein indicating a stoichiometry of one enzyme-tetramer per accessible transporter. The binding was sensitive to changes in pH showing an optimum at around pH 7.0. KCl and NaCl inhibited the binding in a simple dose-dependent manner with Ki of 40 and 20 mM, respectively. The binding was also inhibited by NAD+ with an estimated Ki of 3 mM. ATP, on the other hand, enhanced the binding by up to 3-fold in a dose-dependent manner with an apparent Ka of approximately 6 mM. The binding was not affected by D-glucose or cytochalasin B. The binding did not affect either the glucose or cytochalasin B in binding affinities or the transport activity of the transporter. However, the enzyme was inactivated totally upon binding to the transporter. Based on these findings, we suggest that a significant portion of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes exists as an inactive form via an ATP-dependent, reversible association with glucose transporter, and that this association may exert regulatory intervention on nucleotide metabolism in vitro.

Target size of D-beta-hydroxybutyrate dehydrogenase. Functional and structural molecular weight based on radiation inactivation.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apoenzyme has been purified to homogeneity from beef heart; it is devoid of lipid and inactive. It can be functionally reconstituted with lecithin or phospholipid mixtures containing lecithin. The active form of the enzyme is the enzyme-phospholipid complex. Classical target analysis of radiation-inactivation data has now been used to determine the molecular size of the enzyme both in the native membrane (submitochondrial vesicles) and in the reconstituted enzyme inserted into phospholipid vesicles containing lecithin. For both forms of the enzyme, we find the same molecular size, approximately 110,00 daltons. This size is consistent with a tetramer. Radiation results in fragmentation of the polypeptide and the destruction of the polypeptide correlates with loss of enzymic function. A similar size is obtained when purified D-beta-hydroxybutyrate dehydrogenase is inserted into a nonactivating mixture of phospholipid (i.e. in the absence of lecithin). We conclude that: 1) the native enzyme in submitochondrial vesicles and the purified active enzyme in phospholipid vesicles are the same size, approximating a tetramer; 2) radiation of D-beta-hydroxybutyrate dehydrogenase results in loss of activity and fragmentation of the polypeptide; and 3) the role of lecithin in activation of D-beta-hydroxybutyrate dehydrogenase is unrelated to determining oligomeric size of the enzymes since both active and nonactive forms exhibit the same structural size.