Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.     

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.     

Biotin-conjugated reagents as site-specific probes of membrane protein structure: application to the study of the human erythrocyte hexose transporter

A novel labeling procedure using biotin-conjugated protein-modifying reagents has been employed to study the structure and function of the human erythrocyte hexose transporter. The carbohydrate moiety of the isolated, reconstituted transporter was labeled by using galactose oxidase/biotin hydrazide. Cysteine residues, which are essential for transporter function, were tagged with a biotin-conjugated maleimide. Labeling with this reagent inhibited the binding of cytochalasin B to the transporter. Following sodium dodecyl sulfate-gel electrophoresis, labeling of the transporter and its proteolytic fragments was detected by Western blotting and probing with alkaline phosphatase-conjugated avidin. After tryptic cleavage of the transporter into two membrane domains, preparations reacted with galactose oxidase/biotin hydrazide were labeled on the 25-kDa glycosylated fragment, but not on the carbohydrate-free 19-kDa peptide. Biotin-maleimide-labeled cysteine residues on both peptides. Transporter polypeptide was fragmented more extensively using Staphylococcus aureus V8 protease. Limited digestion produced a broad band of 30-50 kDa and sharper bands of 23 and 21 kDa. More extensive digestion resulted in the disappearance of the 23-kDa peptide and the appearance of sharp bands of 20, 19, 17, 13, 11, 8, and 7 kDa. Biotin label introduced with galactose oxidase/biotin hydrazide was found on the broad 30-kDa band, confirming its identity as a glycopeptide. All of the peptides weighing more than 11 kDa contained cysteine residues labeled with biotin maleimide, while the 8- and 7-kDa peptides were unlabeled. These results demonstrate the potential usefulness of biotin-conjugated reagents as site-specific probes of membrane protein structure.     

Biochemical characterization and subcellular distribution of the glucose transporter from rat brain microvessels

This study describes the biochemical characterization and subcellular distribution of glucose transporters from isolated rat brain cortical microvessels. The D-glucose inhibitable [3H]cytochalasin B binding assay was used to quantitate glucose transporter binding sites in plasma membranes, high-density microsomes and low-density microsomes prepared from basal and insulin-stimulated cells. Incubation with insulin for 30 min increased the number of glucose transporters in the high-density microsomes by around 33% but had no effect on the number of glucose transporters in the plasma membrane or low-density microsomes. Prolonged incubation with insulin (2 h), however, resulted in a small but significant redistribution of glucose transporters to the low-density microsomes. Preincubation of cells with cycloheximide blocked this insulin-induced increase in glucose transporter number, suggesting that this effect of insulin was due to the synthesis of new glucose transport proteins. Specific labeling of glucose transporters was achieved by photoincorporation of [3H]cytochalasin B. Labeled membranes from all fractions contained a single D-glucose inhibitable peak, migrating with a molecular size of 55 kDa on SDS-polyacrylamide gel electrophoresis. Isoelectric focusing of the 55 kDa protein revealed one major peak of D-glucose inhibitable radioactivity focusing at pH 6.0 in all fractions.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

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.     

Molecular characteristics of rat brain glucose transporter: a novel species with Mr 45,000

The glucose transporter of rat brain was examined by the use of cytochalasin B, a potent inhibitor. The dissociation constants (Kd) of D-glucose-inhibitable cytochalasin B binding in various membrane fractions were about 100 nM. Solubilization and partial purification of glucose transporter were carried out by procedures of DE 52 column chromatography, Bio Gel HT column chromatography and Sepharose CL-6B column chromatography from postnuclear membrane fraction. Purified transporter, reconstituted in lipid vesicles, showed D-glucose-specific transport activity with a Michaelis constant (Km) of 7 mM. The molecular weight was estimated to be about 200K by gel filtration in the presence of 0.1% Triton X-100. The subunit molecular weight was estimated to be 45K by SDS-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a covalent probe, indicating that rat brain glucose transporter is a tetramer.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Distribution of the Glucose Transporter in the Mammalian Brain

We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000-64,000 daltons.     

D-Glucose-sensitive and -insensitive cytochalasin B binding proteins from microvillous plasma membranes of human placenta. Identification of the D-glucose transporter

Cytochalasin B was found to bind to at least two distinct sites in human placental microvillous plasma membrane vesicles, one of which is likely to be intimately associated with the glucose transporter. These sites were distinguished by the specificity of agents able to displace bound cytochalasin B. [3H]Cytochalasin B was displaceable at one site by D-glucose but not by dihydrocytochalasin B; it was displaceable from the other by dihydrocytochalasin B but not by D-glucose. Some binding which could not be displaced by D-glucose + cytochalasin B binding site. Cytochalasin B can be photoincorporated into specific binding proteins by ultraviolet irradiation. D-Glucose specifically prevented such photoaffinity labeling of a microvillous protein component(s) of Mr = 60,000 +/- 2000 as determined by urea-sodium dodecyl sulfate acrylamide gel electrophoresis. This D-glucose-sensitive cytochalasin B binding site of the placenta is likely to be either the glucose transporter or be intimately associated with it. The molecular weight of the placental glucose transporter agrees well with the most widely accepted molecular weight for the human erythrocyte glucose transporter. Dihydrocytochalasin B prevented the photoincorporation of [3H]cytochalasin B into a polypeptide(s) of Mr = 53,000 +/- 2000. This component is probably not associated with placental glucose transport. This report presents the first identification of a sodium-independent glucose transporter from a normal human tissue other than the erythrocyte. It also presents the first molecular weight identification of a human glucose-insensitive high-affinity cytochalasin B binding protein.     

Interaction of forskolin with the P-glycoprotein multidrug transporter.

Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug resistance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl] carbamyl]-7-deacetylforskolin, 125I-7-AIPP-Fsk, and 6-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl]carbamyl]forskolin, 125I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and adenylyl cyclase, respectively (Morris et al., 1991). Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. There was no specific labeling of proteins in membranes from the SKOV3 cells. The overexpressed 140-kDa protein in SKVLB membranes was identified as the P-glycoprotein by immunoblot analysis and immunoprecipitation using anti-P-glycoprotein antiserum. Total inhibition of photolabeling of the P-glycoprotein was observed with verapamil, nifedipine, diltiazem, and vinbalastine, and partial inhibition was observed with colchicine and cytochalasin B. Forskolin was less effective at inhibiting the photolabeling of the P-glycoprotein than 1,9-dideoxyforskolin or a lipophilic derivative of forskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of N-glycosylation of GLUT1 for glucose transport activity.

To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.     

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which [43 kilodalton (kDa)] exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an 125I-labeled high-affinity reagent [a triantennary glycopeptide containing an aryl azide group on galactosyl residues; Lee, R. T., & Lee, Y. C. (1986) Biochemistry 25, 6835-6841], the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. We postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work [Hardy, M. R., Townsend, R. R., Parkhurst, S. M., & Lee, Y. C. (1985) Biochemistry 24, 22-28].     

Biochemical and functional heterogeneity of rat adipocyte glucose transporters

We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing of the eluted 40-55-kDa proteins revealed two peaks of D-glucose-inhibitable [3H]cytochalasin B radioactivity focusing at pH 6.4 and 5.6 when low-density microsomes were used as the starting material. In contrast, only one D-glucose inhibitable peak, focusing at pH 5.6, was found in plasma membranes. Pretreatment of the cells with insulin led to a marked redistribution of the pH 5.6 form of the glucose transporter from low-density microsomes to plasma membranes with no effect on the pH 6.4 form of the glucose transporter. Following isolation from the isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels, both glucose transporter isoforms were shown to cross-react with an antiserum raised against the purified human erythrocyte glucose transporter. Following incubation of [3H]cytochalasin B-labeled low-density microsomal and plasma membranes with neuraminidase, the pH 5.6 transporter isoform was shifted on isoelectric focusing to a more basic pH, while the pH 6.4 isoform was not affected. These data demonstrate that: there is a heterogeneity of glucose transporter species in the intracellular pool while the plasma membrane transporters are more uniform in structure. The pH 5.6 glucose transporter isoform is translocated by insulin from the low-density microsomes to the plasma membrane but the pH 6.4 isoform is not sensitive to insulin. Differential sensitivity of the glucose transporter isoforms to neuraminidase suggests that the heterogeneity is at least partially due to differences in glycosylation state.     

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.     

Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule

Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.     

A high molecular weight topoisomerase I from Xenopus laevis ovaries

DNA topoisomerase I has been purified from homogenates of mature Xenopus laevis ovaries. The initial stages in purification of the native enzyme employed a rapid series of three chromatographic steps, followed by gel filtration performed in the presence of sodium dodecyl sulfate. Polypeptides that might represent topoisomerase I were identified by specific labeling of the topoisomerase species with radioactive DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of topoisomerase I radiolabeled with DNA identified three polypeptides with mobilities consistent with sizes of 165, 125, and 88 kDa. All three polypeptides were found to possess topoisomerase activity following elution from the gel and renaturation. Partial proteolytic digestion of the radiolabeled 165-, 125-, and 88-kDa polypeptides with Staphylococcus aureus V8 endoproteinase resulted in identical autoradiographic patterns. This suggests that the 125-kDa and 88-kDa polypeptides may be degradation products of the 165-kDa species. The 165-kDa topoisomerase I exhibited the same sensitivity to camptothecin as the total, native topoisomerase I fraction.     

Identification of a novel Mr = 76-kDa form of beta-adrenergic receptors

The structure of the human beta-adrenergic receptor in purified basal membranes of human placental syncytiotrophoblast was probed using photoaffinity labeling. Basal membranes display a high specific activity of receptors (4-5 pmol/mg protein) and possess both beta 1- and beta 2-adrenergic receptors subtypes. Autoradiography of membranes that were incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, identified four radiolabeled peptides, Mr = 65-kDa, 54-kDa, 43-kDa and a novel higher molecular weight 76-kDa form of the receptor. Photoaffinity labeling of each of these four peptides displayed the pharmacological properties expected for true beta-adrenergic receptors. The 76-kDa photoaffinity labeled receptor peptide observed in human placenta basal membranes has not been reported elsewhere. Competition studies with the beta1-selective ligand CGP-20712A demonstrate that the photoaffinity labeled receptor peptides are composed of both beta 1- and beta 2-adrenergic receptor subtypes.     

Immunological identification of the human erythrocyte monosaccharide transporter

Antibodies to the purified cytochalasin B binding component of the human erythrocyte glucose transporter were prepared in rabbits. They precipitated detergent-solubilized transporter, and partially inhibited its binding of cytochalasin B. The antibodies were used to locate the transporter polypeptide in SDS-polyacrylamide gels of erythrocyte membranes prepared from freshly drawn blood in the presence of protease inhibitors. They labelled only the region of the gel corresponding to that occupied by the purified transporter, with an apparent molecular weight range of 45,000–75,000. These findings indicate that the isolated transporter does not arise by proteolytic degradation of a larger polypeptide, either during the storage of blood or during purification of the transporter.