Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.

Net sugar transport is a multistep process. Evidence for cytosolic sugar binding sites in erythrocytes.

Human erythrocyte net sugar transport is hypothesized to be rate-limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules [Naftalin, R.J., Smith, P.M., & Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249]. The present study examines these hypotheses. Protein-mediated 3-O-methylglucose uptake at 4 degrees C by human erythrocytes and by resealed, hypotonically lysed erythrocytes (ghosts) is inhibited by increasing solvent viscosity. Protein-mediated transport and transbilayer diffusion of the slowly transported substrate 6-NBD glucosamine are unaffected by increasing solvent viscosity. These findings suggest that protein-mediated 3-O-methylglucose transport is diffusion-limited in erythrocytes. More detailed analyses of red cell 3-O-methylglucose uptake (at 4 degrees C and at limiting extracellular sugar levels) reveal that net influx is a biexponential process characterized by rapid filling of a small compartment (C1 = 29 +/- 6% total cell volume; k1 = 7.4 +/- 1.7 min-1) and slow filling of a larger compartment (C2 = 71 +/- 6% total cell volume k2 = 0.56 +/- 0.11 min-1). Erythrocyte D-glucose net uptake at 4 degrees C is also a biphasic process. Transmembrane sugar leakage is a monoexponential process indicating that multicomponent, protein-mediated uptake does not result from sugar uptake by two cell populations of differing cellular volume. Sugar exit at limiting 3-O-methylglucose concentrations is described by single exponential kinetics. This demonstrates that multicomponent sugar uptake does not result from influx into two populations of cells with widely different sugar transporter content. We conclude that biexponential sugar uptake results from slow (relative to transport) exchange of sugars between serial, intracellular sugar compartments. Biexponential sugar uptake is observed under equilibrium exchange conditions (intracellular sugar concentration = extracellular sugar concentration) but only at 3-O-methylglucose concentrations of less than 1 mM. Above this sugar concentration, exchange uptake is a monoexponential process. Because diffusion rates are independent of diffusant concentration, this suggests that multicomponent uptake results from high-affinity sugar binding within the cell. The concentration of cytosolic binding sites (30 microM, Kd(app) = 400 microM) was estimated from the equilibrium cellular 3-O-methylglucose space. Biexponential net 3-O-methylglucose uptake is also observed in human erythrocyte ghosts, in control human K562 cells, and in K562 cells induced to synthesize hemoglobin by prolonged exposure to hemin. This demonstrates that neither membrane-bound nor free cytosolic hemoglobin forms the sugar-binding complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain.

Cytosolic phospholipase A2 (cPLA2) associates with natural membranes in response to physiological increases in Ca2+, resulting in the selective hydrolysis of arachidonyl phospholipids. The isolation and sequence analysis of cPLA2 cDNA clones from four different species revealed several highly conserved regions. The NH2-terminal conserved region is homologous to several other Ca(2+)-dependent lipid-binding proteins. Here we report that the first 178 residues of cPLA2, containing the homologous Ca(2+)-dependent lipid-binding (CaLB) motif, and another recombinant protein containing the cPLA2(1-178) fragment placed at the COOH terminus of the maltose-binding protein (MBP-CaLB) associate with membranes in a Ca(2+)-dependent manner. cPLA2 and MBP-CaLB also bind to synthetic liposomes at physiological Ca2+ concentrations, demonstrating that accessory proteins are not required. In contrast, delta C2, a truncated cPLA2 lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates. However, both delta C2 and cPLA2 hydrolyze monomeric 1-palmitoyl-2-lysophosphatidylcholine at identical rates in a Ca(2+)-independent fashion. These results delineate two functionally distinct domains of cPLA2, the Ca(2+)-independent catalytic domain, and the regulatory CaLB domain that presents the catalytic domain to the membrane in response to elevated Ca2+.

A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP.

We report the cloning and expression of a cDNA encoding a high molecular weight (85.2 kd) cytosolic phospholipase A2 (cPLA2) that has no detectable sequence homology with the secreted forms of PLA2. We show that cPLA2 selectively cleaves arachidonic acid from natural membrane vesicles and demonstrate that cPLA2 translocates to membrane vesicles in response to physiologically relevant changes in free calcium. Moreover, we demonstrate that an amino-terminal 140 amino acid fragment of cPLA2 translocates to natural membrane vesicles in a Ca(2+)-dependent fashion. Interestingly, we note that this 140 amino acid domain of cPLA2 contains a 45 amino acid region with homology to PKC, p65, GAP, and PLC. We suggest that this homology delineates a Ca(2+)-dependent phospholipid-binding motif, providing a mechanism for the second messenger Ca2+ to translocate and activate cytosolic proteins.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter.

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

Cloning and expression of multiple protein kinase C cDNAs.

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.

Molecular cloning of a cDNA encoding human antihaemophilic factor.

A complete copy of the mRNA sequences encoding human coagulation factor VIII:C has been cloned and expressed. The DNA sequence predicts a single chain precursor of 2,351 amino acids with a relative molecular mass (Mr) 267,039. The protein has an obvious domain structure, contains sequence repeats and is structurally related to factor V and ceruloplasmin.

Transmembrane location of oligosaccharide-lipid synthesis in microsomal vesicles.

The oligosaccharide-lipid which is the precursor of asparagine-linked oligosaccharides of eucaryotic glycoproteins is synthesized from sugar nucleotides in the endoplasmic reticulum. The transmembrane location of the assembly of this oligosaccharide-lipid has been studied in vitro in rat liver microsomes. Protease treatment of these sealed vesicles which are derived from the endoplasmic reticulum resulted in the inactivation of a number of enzymes of oligosaccharide-lipid synthesis. Three early steps, the synthesis of dolichol--phosphate--mannose, of dolichol--phosphate--glucose and of dolichol--pyrophosphoryl--di--N--acetylchitobiose, as well as the final steps, the addition of glucose residues to oligosaccharide-lipid, were inactivated under conditions where only the cytoplasmic side of the membrane was accessible to protease. This finding, and the fact that no activities were latent to protease in intact microsomal vesicles, suggest that oligosaccharide-lipid is assembled on the cytoplasmic side of the microsomal membrane. However, the possibility of enzymes spanning the bilayer with their active sites facing the lumen cannot be ruled out. These results are discussed in relation to the segregation of newly made glycoprotein products within the lumen of the endoplasmic reticulum.