Characterization and partial purification of liver glucose transporter GLUT2.
(33/2418)
GLUT2, the major facilitative glucose transporter isoform expressed in hepatocytes, pancreatic beta-cells, and absorptive epithelial cells, is unique not only with its low affinity and broad substrate specificity as a glucose transporter, but also with its implied function as a glucose-sensor. As a first essential step toward structural and biochemical elucidation of these unique, GLUT2 functions, we describe here the differential solubilization and DEAE-column chromatography of rat hepatocyte GLUT2 protein and its reconstitution into liposomes. The reconstituted GLUT2 bound cytochalasin B in a saturable manner with an apparent dissociation constant (K(d)) of 2.3 x 10(-6) M and a total binding capacity (B(T)) of 8.1 nmol per mg protein. The binding was completely abolished by 2% mercury chloride, but not affected by cytochalasin E. Significantly, the binding was also not affected by 500 mM D-glucose or 3-O-methyl D-glucose (3OMG). The purified GLUT2 catalyzed mercury chloride-sensitive 3OMG uptake, and cytochalasin B inhibited this 3OMG uptake. The inhibition was dose-dependent with respect to cytochalasin B, but was independent of 3OMG concentrations. These findings demonstrate that our solubilized GLUT2 reconstituted in liposomes is at least 60% pure and functional, and that GLUT2 is indeed unique in that its cytochalasin B binding is not affected by its substrate (D-glucose) binding. Our partially purified GLUT2 reconstituted in vesicles will be useful in biochemical and structural elucidation of GLUT2 as a glucose transporter and as a possible glucose sensor. (+info)
Hyaluronan exists in the normal stratum corneum.
(34/2418)
Hyaluronan is well known to exist as a water-sorbed macromolecule in the extracellular matrix. We here examined whether hyaluronan exists in the normal stratum corneum. High performance liquid chromatography was used to quantify hyaluronan content in the stratum corneum, epidermis (including stratum corneum), and dermis of mice, with the resulting dry weights being 22.3 +/- 2.9, 15.1 +/- 1.5, and 738.6 +/- 31.6 microg per g, respectively. Normal mouse skin was then labeled with [3H]-glucosamine in an organ culture, and accumulation of [3H]-labeled hyaluronan and its molecular mass were determined separately for the stratum corneum, epidermis, and dermis. In the stratum corneum, [3H]-labeled hyaluronan was accumulated linearly over the 3-d culture period. After the 3-d culture period, the epidermis synthesized twice the amount (expressed as dpm per mg dry weight) of [3H]-labeled hyaluronan as the dermis, whereas the stratum corneum and dermis showed nearly the same content of [3H]-labeled hyaluronan. The molecular mass of [3H]-labeled hyaluronan was highest (>1.0 x 106) in the dermis and clearly lower (<6.0 x 104) in the stratum corneum. Based on these results, we here confirm that hyaluronan is supplied from keratinocytes beneath the stratum corneum layer, and is present in the normal stratum corneum. We speculate that hyaluronan may play a role in moisturizing the stratum corneum and/or regulating its mechanical properties. (+info)
Characterization of serum factors modulating splenic cytotoxicity in a syngeneic rat tumour system.
(35/2418)
During the terminal stages of tumour growth (6-8 weeks) in Wistar rats bearing a syngeneic squamous cell carcinoma (Sp1), their sera can block in vitro anti-tumour cytotoxicity by immune splenic T lymphocytes. At an earlier stage of tumour growth (4-6 weeks) the sera do not block this cytotoxicity, but can induce anti-tumour cytotoxicity by non-immune spleen cells in the absence of complement. Sera taken at these 2 stages of tumour growth have been fractionated by ion-exchange chromatography, using DEAE-cellulose. The fractions have been examined by immunoelectrophoresis and tested for anti-tumour reactivity. Blocking activity was found in the Week-8 serum fraction eluted with 0-005M phosphate buffer, pH 7-4, whilst the "cytotoxic" activity of Week-4 serum was eluted with 0-02M phosphate buffer, pH 6-2. It is suggested that different IgG sub-classes are responsible for the 2 activities. (+info)
Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.
(36/2418)
Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed. (+info)
Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase.
(37/2418)
The DNA-directed in vitro synthesis of beta-galactosidase has been investigated in a system dependent on Escherichia coli ribosomes, a salt wash of the ribosomes, and a supernatant fraction. Fractionation of the supernatant has made it possible to obtain dependencies on RNA polymerase and another protein factor for beta-galactosidase synthesis. The other factor (called L factor) cannot be replaced by a variety of proteins known to be required for transcription and translation. It has been purified to homogeneity and has a molecular weight of approximately 65,000. Although it is required for the in vitro synthesis of beta-galactosidase, it has no effect on total DNA-dependent amino acid incorporation under the conditions of the incubation. However, total RNA synthesis is depressed by the addition of L factor in a manner similar to what is observed with rho factor could not replace L factor in beta-galactosidase synthesis. (+info)
Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli.
(38/2418)
Two strains of Escherichia coli were inhibited by complement-inactivated cow serum and to a lesser extent by precolostral calf serum devoid of specific antibodies. They were not inhibited by undiluted colostral whey or milk but colostral whey became bacteriostatic after dialysis or dilution in Kolmer saline and addition of precolostral calf serum or lactoferrin. The inhibition in all these fluids was due to iron-binding proteins (transferrin or lactoferrin). Undiluted dialysed milk was not inhibitory because of its low content of lactoferrin but became inhibitory after addition of 1 mg/ml of lactoferrin. The lack of inhibition in undiluted whey is due to the high concentration of citrate in colostral whey (and milk) and it is suggested that citrate competes with the iron-binding proteins for iron and makes it availabe to the bacteria. Addition of bicarbonate, which is required for the binding of iron by transferrin and lactoferrin, can overcome the effect of citrate; hence, the bacteriostatic effect of cow serum and precolostral calf serum is due to the presence of both transferrin and bicarbonate as well as the low lefel of citrate. (+info)
Molecular identification of oligoalginate lyase of Sphingomonas sp. strain A1 as one of the enzymes required for complete depolymerization of alginate.
(39/2418)
A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster. (+info)
Red cell proteins. I. Two-dimensional mapping of human erythrocyte lysate proteins.
(40/2418)
Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants. (+info)