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At least 19 recordsLinked to original sources
PubMed1989

Visualization of membrane-associated folate transport proteins.

Transport of Methotrexate (MTX) into cells, via the "reduced folate" transport system, is a critical factor in the effectiveness of the drug in cancer chemotherapy, and defective transport is one of the principal types of resistance to MTX. Probes capable of detecting membrane-associated folate transport proteins (ftp's) in individual cells are potentially useful for identifying structural and functional domains and for investigating mechanisms of substrate translocation. Polyclonal antibody to highly purified ftp from Lactobacillus casei, in conjunction with a second, gold-labeled antibody, has been used to visualize, via electron microscopy, the protein in Triton-treated membrane fragments and in the membrane and cytoplasm of spheroplasts. To visualize ftp in L1210 cells, the substrate-binding site was first labeled covalently with activated fluorescein-Methotrexate, and the cells were then treated with anti-fluorescein antibody and the gold-labeled antibody.

Animals↗
PubMed1977

Similar polypeptide composition of fast-transported proteins in rat motor and sensory axons.

SDS-polyacrylamide gel electrophoresis was used to characterize labeled proteins transported in rat motor and sensory axons after application of 3H-leucine to the neuron cell bodies. Two types of experiments were performed: first, transported protein accumulating proximal to a ligature placed on the sciatic nerve was analyzed; second, the segment of sciatic nerve nearest to the "wavecrest" of transported protein travelling down the nerve was analyzed. In both cases, no significant differences in peak position or amplitude were found in gels containing labeled proteins from motor or sensory axons. This may mean that the majority of fast-transported protein is involved in an axonal function common to the two types of neuron.

Animals↗
PubMed1989

[Changes in the axonal protein transport under conditions of elevated functional activity of the neurons].

The prolonged nonintensive physical activity by swimming without load (12 +/- 2 h.) has no effect on the overall amount of fast and slow transported proteins of transport velocity in rat central and peripheral sensory fibres of the sciatic nerve. However, the rate of fast axonal transport in the motor fibres decreases by 18% and the amount of proteins by a factor of 2 as compared with control. The rate of slow axonal transport does not change, but the mean level of transported labeled proteins decreases by 1.9 times. The relatively short-term but more intensive activity (swimming with the load during 60 +/- 10 min.) provokes an increase of the rate by 10% and the overall amount of fast transported proteins by 2 times. The rest of the animals during 6 h. returns the above parameters to control values. A suggestion is made that the rate and the amount of transported proteins depend on the variations in functional state of the neurons and their axons.

Animals↗
PubMed1992

Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties.

The structural and functional roles of the single asparagine (N)-linked oligosaccharide chain of Band 3 (AE1), the anion transport protein of the human erythrocyte membrane, were examined. Purified Band 3 (M(r) = 95,000) in 0.1% octaethylene glycol mono n-dodecyl ether (C12E8) detergent solution was deglycosylated using N-glycosidase F. This treatment sharpened the protein band on sodium dodecyl sulfate gel electrophoresis and decreased its apparent molecular weight by 5,000. The purified membrane domain could be deglycosylated under similar conditions, causing a shift from a broad band centered at 55 kDa to a sharp 46-kDa band. Band 3 was shown to bind tomato lectin, and loss of lectin binding on blots provided a sensitive assay for deglycosylation. Carbohydrate analysis revealed that greater than 80% of the oligosaccharide could be removed from Band 3 by N-glycosidase F digestion. The deglycosylated protein maintained its dimeric structure and level of detergent binding but had a smaller Stokes radius (RS = 72 A) than native Band 3 (RS = 75 A). The Stokes radius of the membrane domain (RS = 60 A) also decreased upon deglycosylation (RS = 58 A). Circular dichroism studies showed that deglycosylation did not change the secondary structure of Band 3 or the membrane domain. The sensitivity of Band 3 or the membrane domain to proteolytic digestion by trypsin or proteinase K was also unaffected by deglycosylation. The deglycosylated protein aggregated more rapidly and was much more readily precipitable by ammonium sulfate. The deglycosylated protein bound the anion transport inhibitor 4-benzamido-4'-amino-stilbene-2,2'-disulfonate with the same affinity (Kd = 1 microM) as the native protein. Transport studies using reconstituted Band 3 and resealed ghosts showed that deglycosylated Band 3 retained its ability to transport anions. We conclude that removal of the oligosaccharide chain from Band 3 and any resultant structural changes had no effect on the transport function of this protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1992

A kinetic study of the role of band 3 anion transport protein in the transport of salicylic acid and other hydroxybenzoic acids across the human erythrocyte membrane.

The mechanism of transport of salicylic acid and five other hydroxybenzoic acids across the human erythrocyte membrane was investigated. The specific anion transport inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, reduced but did not totally abolish transport of these acids. This observation suggests that these acids are transported by two parallel processes, one involving the band 3 anion transport protein channel and the other probably involving passive diffusion of the un-ionized molecule. The activation energies for membrane transport were large (99-127 kJ.mol-1), an observation that is consistent with the rate-limiting step for anion transport being the conformational change of the band 3 anion transport protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucose-transport protein GLUT 2.

Antibodies raised against a 52 kDa rat liver microsomal glucose-transport protein were used to screen a rat liver cDNA library. Six positive clones were isolated. Two clones were found to be identical with the liver plasma-membrane glucose-transport protein termed GLUT 2. The sequence of the four remaining clones indicates that they encode a unique microsomal facilitative glucose-transport protein which we have termed GLUT 7. Sequence analysis revealed that the largest GLUT 7 clone was 2161 bp in length and encodes a protein of 528 amino acids. The deduced amino acid sequence of GLUT 7 shows 68% identity with the deduced amino acid sequence of rat liver GLUT 2. The GLUT 7 sequence is six amino acids longer than rat liver GLUT 2, and the extra six amino acids at the C-terminal end contain a consensus motif for retention of membrane-spanning proteins in the endoplasmic reticulum. When the largest GLUT 7 clone was transfected into COS 7 cells the expressed protein was found in the endoplasmic reticulum and nuclear membrane, but not in the plasma membrane. Microsomes isolated from the transfected COS 7 cells demonstrated an increase in their microsomal glucose-transport capacity, demonstrating that the GLUT 7 clone encodes a functional endoplasmic-reticulum glucose-transport protein.

Amino Acid Sequence↗
PubMed1991

Monomeric erythrocyte band 3 protein transports anions.

The anion transport system of the human erythrocyte membrane was reconstituted in egg phosphatidylcholine membranes by using either the unmodified transport protein, band 3, or covalently crosslinked band 3 dimers. Unilamellar vesicles of a diameter of 32 +/- 3 nm were then isolated from the sample by passage through a French press and subsequent gel filtration. According to sedimentation equilibrium measurements, around 85% of the vesicles were devoid of protein. The remaining 15% contained either a single band 3 monomer or, when crosslinked band 3 protein was used, a single band 3 dimer. Vesicles containing either single monomers or single dimers showed a rapid, inhibitor-sensitive sulfate efflux, and the turnover numbers of band 3 for the inhibitor-sensitive flux component were identical in both systems. This shows that monomeric band 3 protein is able to transport anions and that dimerization of the protein does not change its transport activity.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1990

Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli.

Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 10----glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.

Alleles↗
PubMed1992

Mammalian nitrobenzylthioinosine-sensitive nucleoside transport proteins. Immunological evidence that transporters differing in size and inhibitor specificity share sequence homology.

Polyclonal antibodies were raised against the nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter of human erythrocyte membranes. On Western blots of these membranes they labeled the broad "band 4.5" region (average apparent M(r) 55,000), which contains both the nucleoside and glucose transport proteins. However, they did not recognize the glucose transporter when this was prepared free of nucleoside transporter by expression from a cDNA clone. Their specificity for the nucleoside transporter was confirmed by the ability to immunoadsorb NBMPR- but not cytochalasin B-binding sites from a detergent-solubilized mixture of band 4.5 proteins. Although a large proportion of the antibodies recognized extracellular epitopes, these appeared to be located primarily on the polypeptide moiety of the glycoprotein, as demonstrated by the ability of the antibodies strongly to label the deglycosylated transporter (apparent M(r) 45,000) on Western blots. The antibodies were species-cross-reactive, recognizing nucleoside transporters from pig and rabbit erythrocytes and from rat liver. The pig protein is similar to the human transporter in its inhibitor sensitivity but is considerably larger (apparent M(r) 57,000 after deglycosylation). In contrast, the rat protein is similar in size to the human transporter (apparent M(r) 45,000 after deglycosylation) but much less sensitive to the inhibitors dilazep and dipyridamole. These findings indicate that despite their differences in size and inhibitor specificity, the NBMPR-sensitive nucleoside transporters of these mammalian species are related in amino acid sequence.

Affinity Labels↗
PubMed1991

Analysis of protein transport through the Golgi in a reconstituted cell-free system.

The processes which transport membrane proteins between compartments of the Golgi apparatus have been reconstituted in vitro using isolated Golgi fractions. This cell-free system allows a detailed analysis of protein transport not possible in intact cells. Transport of the membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is measured from a "donor" to an "acceptor" Golgi fraction. The donor Golgi fraction is prepared from VSV-infected Chinese hamster ovary (CHO) mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase I. "Acceptor" is prepared from uninfected wild-type CHO cells. Transport is measured by the addition of N-acetylglucosamine to G protein, which can occur only upon movement of G protein from donor to acceptor. Transport requires physiological pH and osmolarity, is dependent on nucleotide triphosphates, and is mediated by proteins both from cytosol and on the Golgi membranes. Protein movement is inhibited by the non-hydrolyzable GTP analogue, GTP gamma S. The process of transport proceeds through the budding, pinching off, targeting, and fusion of transport vesicles. In this system these vesicles are initially coated with a non-clathrin coat and are targeted with this coat intact. Several of the proteins which mediate transport have been characterized, and isolated to homogeneity. The successful development of this assay has led to the formulation of cell free assays for protein transport between other compartments. Comparison of these systems indicates that some common mechanisms of vesicular movement are used in transport between a variety of membrane compartments.

Animals↗
PubMed1989

Retinol-binding protein: the serum transport protein for vitamin A.

The information available regarding the chemical structure of RBP, the structure of the RBP gene, and the expression of the RBP gene has expanded dramatically in recent years. Still many questions concerning RBP remain to be answered. The longstanding and important questions concerning the possible existence and the biochemical characteristics of the RBP cell surface receptor are in need of resolution. The factors that regulate RBP secretion from the liver still remain to be fully elucidated. Additional information concerning the physiological role of RBP synthesis in extrahepatic tissues is needed. Considering what is now known, it is clear that in the future much intense research will be required before the many important questions regarding the structure, synthesis, secretion, and physiological roles of RBP can be answered.

Animals↗
PubMed1992

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.

Amino Acid Sequence↗
PubMed1990

Glucose transporter protein content and glucose transport capacity in rat skeletal muscles.

The relationships among fiber type, glucose transporter (GLUT-4) protein content, and glucose transport activity stimulated maximally with insulin and/or contractile activity were studied by use of the rat epitrochlearis (15% type I-20% type II2a-65% type IIb), soleus (84-16-0%), extensor digitorum longus (EDL, 3-57-40%), and flexor digitorum brevis (FDB, 7-92-1%) muscles. Insulin-stimulated 2-deoxy-D-glucose (2-DG) uptake was greatest in the soleus, followed (in order) by the FDB, EDL, and epitrochlearis. On the other hand, contractile activity induced the greatest increase in 2-DG uptake in the FDB, followed by the EDL, soleus, and epitrochlearis. The effects of insulin and contractile activity on 2-DG uptake were additive in all the muscle preparations, with the relative rates being FDB greater than soleus greater than EDL greater than epitrochlearis. Quantitation of the GLUT-4 protein content with the antiserum R820 showed the following pattern: FDB greater than soleus greater than EDL greater than epitrochlearis. Linear regression analysis showed that whereas a relatively low and nonsignificant correlation existed between GLUT-4 protein content and 2-DG uptake stimulated by insulin alone, significant correlations existed between GLUT-4 protein content and 2-DG uptake stimulated either by contractions alone (r = 0.950) or by insulin and contractions in combination (r = 0.992). These results suggest that the differences in maximally stimulated glucose transport activity among the three fiber types may be related to differences in their content of GLUT-4 protein.

Actins↗
PubMed1992

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.

Adenosine Triphosphate↗
PubMed1990

Effect of target removal on goldfish optic nerve regeneration: analysis of fast axonally transported proteins.

How is axonal transport in regenerating neurons affected by contact with their synaptic target? We investigated whether removing the target (homotopic) lobe of the goldfish optic tectum altered the incorporation of 3H-proline into fast axonally transported proteins in the regenerating optic nerve. Regeneration was induced either by an optic tract lesion (to reveal the changes in the original axon segment that remained connected to the cell body) or by an optic nerve lesion (to reveal the changes in the newly formed axon segment). Of 26 proteins analyzed by 2-dimensional gel electrophoresis and fluorography, all but one showed increased labeling as a result of tectal lobe ablation. By 2 d after the lesion, significantly increased labeling of some proteins was seen with a 6-hr labeling interval, but not with a 24-hr labeling interval. This is probably indicative of an increased velocity of transport, which may have been a nonspecific consequence of the surgery. Otherwise, tectal lobe removal had relatively little effect until 3 weeks, when there was a transitory increase in labeling of transported proteins in the new axon segments of the tectum-ablated animals. Beginning at 5 weeks, tectal lobe ablation caused considerably higher labeling of many of the proteins in the original axon segments. Because this was seen with both 6-hr and 24-hr labeling intervals, it is probably indicative of increased protein synthesis. The increased synthesis lasted until at least 12 weeks, though some proteins were beginning to show a diminished effect at this time. In the late stages of regeneration (8-12 weeks), there was also increased labeling of proteins in the new axon segments as a result of the absence of the target tectal lobe. This included a disproportionately large increase in the relative contribution of cytoskeletal proteins and of protein 4, which is the goldfish equivalent of the growth-associated protein GAP-43 (neuromodulin). We conclude that, after the regenerating axons begin to innervate the tectum, the expression of most of the proteins in fast axonal transport is down-regulated by interaction between the axons and their target. However, the changes in expression may be preceded by a modulation of the turnover and/or deposition of proteins in the newly formed axon segment.

Animals↗
PubMed1992

Growth cones of regenerating adult sciatic sensory axons release axonally transported proteins.

Labelled, rapidly transported axonal proteins were shown to be released from adult frog sciatic sensory neurons, regenerating in vitro after a crush injury. The spatial distribution of the transported, released proteins could accurately be resolved by culturing the nerve on nitrocellulose paper, which trapped the released proteins. The release was located to the crush and to the entire outgrowth region. When regeneration was inhibited by adenosine, the release was limited to the crush site, implying that the release was linked to the growing axons. Other experiments suggested that the release emanated from growth cones. Furthermore, two-dimensional electrophoretical analysis of both fast axonally transported and of released proteins showed that the latter represented a selection of the transported protein species.

Adenosine↗