Interaction of the 56,000-dalton phosphoprotein phosphatase from reticulocytes with regulin and inhibitor 2

The interaction of divalent metal ions with a homogeneous 56,000-dalton phosphoprotein phosphatase isolated from rabbit reticulocytes was studied. The effects of the ions on enzymatic activity and on fluorescence from a 3-(4-maleimidylphenyl)-4-methyl-7-(diethylamino)coumarin derivative of the protein were compared. Enzymatic activity is dependent on Mn2+. The apparent association constant for Mn2+ is about 0.5 mM-1 as judged from enzymatic activity and from changes in fluorescence caused by binding of the metal ion; Ca2+ and Mg2+ do not affect enzymatic activity and appear not to bind tightly to the enzyme; however, Co2+, Fe2+, and Zn2+ bind to the protein and inhibit the Mn2+-activated enzyme. The 56,000-dalton phosphoprotein phosphatase was found to interact with regulin, a spectrin-associated protein also isolated from reticulocytes, and with skeletal muscle phosphatase inhibitor 2. The interaction was followed by changes in the enzymatic activity and by quenching of fluorescence from the coumarin derivative of the phosphatase. Homogeneous regulin (Mr approximately 230,000) increases the activity of the enzyme severalfold; this stimulation is Mn2+-dependent. Inhibitor 2 decreases enzyme activity but only if the two proteins are preincubated in the absence of Mn2+. Comparable differences in the effect of Mn2+ were also observed in parallel experiments in which changes in fluorescence from the coumarin-labeled 56,000-dalton phosphatase were measured. In these experiments, it was shown that Mn2+ enhances the interaction between regulin and the 56,000-dalton phosphatase, but inhibits the interaction between the phosphatase and inhibitor 2.     

Partial characterization of a 230,000-dalton reticulocyte protein and peptides derived from it that affect the activity of a protein phosphatase

Monoclonal antibodies were raised that recognize a series of highly antigenic, protease-sensitive peptides that modulate protein phosphatase activity in reticulocyte extracts. Purified antigen peptides cause a 3-fold increase in the enzymatic activity of a homogeneous Mr congruent to 56,000 protein phosphatase. The monoclonal antibodies inhibit protein phosphatase activity in crude extracts but do not recognize the protein phosphatase itself. The antigen peptides are associated with the phosphatase throughout its purification from the postribosomal supernatant of rabbit reticulocytes but are separated from it during size exclusion high performance liquid chromatography (see accompanying article: Wollny, E., Watkins, K., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492). The series of antigenic peptides appears to be derived by proteolysis from a 230,000-Da precursor, which is relatively abundant in undegraded form in the membrane fraction of rabbit reticulocytes and is present in erythrocyte ghosts. Antigen peptides are extracted with spectrin from both sources. The Mr congruent to 230,000 peptide is not the alpha or beta subunit of spectrin or ankyrin and appears not to have been recognized previously. The name "regulin" is proposed.     

The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products

Three forms of protein phosphatase-1 were isolated from rabbit skeletal muscle that had Mr values of 37 000, 34 000 and 33 000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the beta-subunit of phosphorylase kinase very much faster than the alpha-subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase-3 and Mg-ATP for activity by incubation with inhibitor-2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr-37 000 and Mr-34 000 species were converted to the Mr-33 000 form by incubation with chymotrypsin. It is concluded that the Mr-33 000 and Mr-34 000 forms are derived from the Mr-37 000 component by limited proteolysis. Conversion of the Mr-37 000 to the Mr-33 000 form was accompanied by a two-fold increase in activity, indicating that an Mr-4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36 000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr-37 000 (15-20 kU/mg) or Mr-33/34 000 (40-50 kU/mg) forms of protein phosphatase-1. It dephosphorylated the alpha-subunit of phosphorylase kinase 4-5-fold faster than the beta-subunit, was unaffected by inhibitor-1 or inhibitor-2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase-3 and Mg-ATP-dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr-37 000 form of protein phosphatase-1 to the Mr-33 000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observed with protein phosphatase 1. Antibody to protein phosphatase-2A raised in sheep did not cross-react with any of the forms of protein phosphatase-1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase-1 and protein phosphatase-2A are distinct gene products.     

Conditioned media of Kupffer and endothelial liver cells influence protein phosphorylation in parenchymal liver cells. Involvement of prostaglandins.

The possible role of Kupffer and endothelial liver cells in the regulation of parenchymal-liver-cell function was assessed by studying the influence of conditioned media of isolated Kupffer and endothelial cells on protein phosphorylation in isolated parenchymal cells. The phosphorylation state of three proteins was selectively influenced by the conditioned media. The phosphorylation state of an Mr-63,000 protein was decreased and the phosphorylation state of an Mr-47,000 and an Mr-97,000 protein was enhanced by these media. These effects could be mimicked by adding either prostaglandin E1, E2 or D2. Both conditioned media and prostaglandins stimulated the phosphorylase activity in parenchymal liver cells, suggesting that the Mr-97,000 phosphoprotein might be phosphorylase. Parenchymal liver cells secrete a phosphoprotein of Mr-63,000 and pI 5.0-5.5. The phosphorylation of this protein is inhibited by Kupffer- and endothelial-liver-cell media, and prostaglandins E1, E2 and D2 had a similar effect. The data indicate that Kupffer and endothelial liver cells secrete factors which influence the protein phosphorylation in parenchymal liver cells. This forms further evidence that products from non-parenchymal liver cells, in particular prostaglandin D2, might regulate glucose homoeostasis and/or other specific metabolic processes inside parenchymal cells. This stresses the concept of cellular communication inside the liver as a way by which the liver can rapidly respond to extrahepatic signals.     

Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme

Pigment-binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene-treated cells as well as in homologous and heterologous cell-free translation systems by isolated polysomes. It is shown that the pigment-binding polypeptides of the light-harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene-treated cells. The large Mr-28 000 polypeptide of the reaction center and the Mr-10 000 pigment-binding polypeptide of the light-harvesting complex II were found to be synthesized by free (water-soluble) polysomes without a cleavable 'leader' or 'signal' peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23-45]. The Mr-10 000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane-assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic-solvent-soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr-10 000 polypeptide may enter the lipid bilayer by a 'membrane-triggered' conformational change.     

Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes.

A method is described for preparing a plasma-membrane fraction from hepatocytes by a rapid, gentle, Percoll fractionation procedure. Cholera toxin elicited the ribosylation of a number of proteins in these membranes, including the components of the stimulatory guanine nucleotide regulatory protein, Ns. Insulin, however, inhibited the ability of cholera toxin to ribosylate a protein of Mr 25 000. The action was decreased in membranes from cells that had been pre-treated with glucagon. Ribosylation of both the components of Ns and the Mr-25 000 species occurred in whole cells treated with cholera toxin, because membranes from such treated cells exhibited decreased labelling when incubated with [32P]NAD+ and activated cholera toxin. The labelling of proteins, including the Mr-25 000 species, with [32P]NAD+ and cholera toxin in the plasma membranes was decreased by an inhibitor of ribosylation. Azido-GTP photoaffinity labelling identified several high-affinity GTP-binding proteins, including one of Mr 25 000. Cholera toxin failed to ribosylate the Mr-25 000 protein in membranes from cells that had been pre-treated with the tumour-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA). In membranes from such treated cells, insulin actually allowed cholera toxin to label this species. As TPA activates protein kinase C, it is possible that the Mr-25 000 protein, or a species that interacts with it, is a substrate for phosphorylation. These observations may offer an explanation for some of the perturbing effects that TPA exerts on insulin's action. It is suggested that the insulin receptor interacts with the guanine nucleotide regulatory protein system in the liver, and that the Mr-25 000 species may be a component of Nin, a specific guanine nucleotide regulatory protein that has been proposed to mediate certain of the actions of insulin on target cells [Houslay & Heyworth (1983) Trends Biochem. Sci. 8, 449-452].     

Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.     

Friend erythroleukemia cell membrane transferrin receptors

We have compared the uptake of transferrin by murine Friend erythroleukemia cells with the uptake of transferrin by murine reticulocytes. Friend cells which had been induced to erythroid differentiation by dimethyl sulfoxide took up transferrin in a manner qualitatively and quantitatively similar to the uptake of transferrin by reticulocytes, while uninduced Friend cells took up only negligible amounts of transferrin. Specific transferrin-binding activity could be demonstrated in detergent extracts of membranes from induced cells and this activity was isolated from membrane extracts by the use of antibody to transferrin. The isolated membrane component(s) with transferrin-binding activity migrated electrophoretically as a single protein on sodium dodecyl sulfate gels and had similar properties to a transferrin-binding protein isolated previously from reticulocytes.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii.

We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii. The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms. The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6. The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM. The subunit Mr of Chlamydomonas ASL is approx. 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000. Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro. In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity. Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver.     

Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.     

Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man

Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis.     

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.     

Insulin stimulates proteolysis of the alpha-subunit, but not the beta-subunit, of its receptor at the cell surface in rat liver.

Insulin receptors in rat liver plasma membranes contain two alpha- and two beta-subunits held together by interchain disulphide bonds ([alpha beta]2 receptors). Affinity-labelled receptors were digested with chymotrypsin or elastase and then exposed to dithiothreitol before solubilization from membranes and SDS/polyacrylamide-gel electrophoresis. This resulted in partial reduction and isolation of Mr-225,000 alpha beta, Mr-200,000 alpha 1 beta, Mr-165,000 alpha beta 1 and Mr-145,000 alpha 1 beta 1 receptor halves containing intact (alpha, beta) or degraded (alpha 1, beta 1) subunits. The ability to identify half-receptor complexes containing intact or degraded subunits made it possible to assay each subunit simultaneously for insulin-induced proteolysis in isolated plasma membranes or during perfusion of rat liver in situ with insulin. In liver membranes, insulin binding increased the fraction of receptors containing degraded alpha-subunits to about one-third of the total population during 2 h of incubation at 23 degrees C. beta-Subunit proteolysis increased only minimally during this time. Plasma membranes isolated from livers perfused with insulin at 37 degrees C contained degraded alpha-subunits but only intact beta-subunits, showing that insulin induced cell-surface proteolysis of the binding, but not the kinase, domain of its receptor. Since previous observations [Lipson, Kolhatkar & Donner (1988) J. Biol. Chem 263, 10495-10501] have shown that receptors containing degraded alpha-subunits are internalized but do not recycle, it is possible that cell-surface degradation may play a role in the regulation of insulin-receptor number in hepatic tissue. Proteolysis of the beta-subunit is not a likely mechanism by which receptor-kinase activity may be attenuated under physiological conditions.     

The insulin- and glucagon-stimulated ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity.

The hormone-stimulated 'dense-vesicle' cyclic AMP phosphodiesterase was solubilized as a proteolytically 'clipped' species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native 'dense-vesicle' enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified 'clipped' enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].     

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.     

Differential sensitivity of reticulocytes to nicked and unnicked diphtheria toxin

The effects of diphtheria toxin (DT) on rabbit reticulocytes were studied. Protein synthesis of the cells was inhibited at relatively high DT concentrations. Several drugs including metabolic inhibitors, nucleotides and amines blocked DT-induced toxicity in reticulocytes in a manner comparable to their known protective effects in other cell lines. This suggested that receptor-mediated binding and internalization of DT occurred in reticulocytes. Highly purified unnicked DT did not inhibit protein synthesis, while nicked toxin was effective. Treatment of the unnicked toxin with trypsin converted it into a potent form. It is hypothesized that reticulocytes lack the required toxin-activating (nicking) enzyme or that the putative enzyme is not located on or in the cell in a manner where it can interact with the toxin.     

GLUTX1, a novel mammalian glucose transporter expressed in the central nervous system and insulin-sensitive tissues

Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.     

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.