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M A Hediger

Publications and source records attributed to M A Hediger.

124 records · Page 7Linked to original sources
PubMed1989

Molecular genetics of the human Na+/glucose cotransporter.

Recent success in expression cloning has revealed the primary structure of the Na+/glucose cotransporter from rabbit small intestine, and this has subsequently led to the cloning of the Na+/glucose cotransporters from human small intestine and human kidney. Close homology is evident between the rabbit and human intestinal Na+/glucose cotransporters at the DNA level, and the predicted amino acid and secondary structure levels. The Na+/glucose cotransporter amino acid sequence from human kidney is 57% identical with that from human small intestine. Significant homology also exists between these Na+/glucose cotransporters and the E. coli Na+/proline cotransporter (putP). The rabbit intestinal Na+/glucose cotransporter has 11 potential membrane spanning regions and 2 hydrophilic regions containing highly charged residues. The amino acid sequence shows two potential N-glycosylation sites (N-X-T/S). Using an in vitro translation approach we were able to determine that only one of these (Asn 248) is glycosylated. Expression experiments with Xenopus oocytes using the N-glycosylation inhibitor tunicamycin indicate that glycosylation of Asn 248 is required for functional expression of the transporter. The N-X-T/S sequence at Asn 248 is conserved in the human intestinal and the human renal Na+/glucose cotransporter. Chromosomal localization studies map the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22 and the human renal Na+/glucose cotransporter gene (SGLT2) to the q-arm of chromosome 16.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine.

The Na+/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed in Xenopus oocytes. Injection of cloned RNA into oocytes increased Na+/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed in Xenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of 14C-alpha-methyl-D-glucopyranoside uptake into brush border membrane vesicles and Xenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na+-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (Ki 6 and 9 microM), and saturable (alpha-methyl-D-glucopyranoside Km 110 and 570 microM). The sugar specificity was examined by competition experiments, and in both cases the selectivity was D-glucose greater than alpha-methyl-D-glucopyranoside greater than D-galactose greater than 3-O-methyl-D-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na+/glucose cotransporter.

Animals↗
PubMed1989

Assignment of the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22.

The chromosomal location of the human intestinal Na+/glucose cotransporter gene (SGLT1) was determined using human cDNA and genomic probes for this transporter gene. Southern blot analysis of genomic DNA from 15 mouse-human somatic cell hybrids showed that the human gene for this transporter resides on chromosome 22. Analysis of hamster-human hybrids selectively retaining chromosome 22 or a portion of it allowed specific assignment of the locus to the q11.2----qter region of chromosome 22. A restriction fragment length polymorphism was identified with EcoRI.

Animals↗
PubMed1989

Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters.

Cotransport proteins are responsible for the active accumulation of organic substrates in cells. Na+ gradients provide the driving force for uptake of most substrates into eukaryotes and for a few substrates in some prokaryotes. We report here the cloning and sequencing of the human intestinal Na+/glucose cotransporter (SGLT1) and compare its structure with other cloned transporters. At the DNA level and the predicted amino acid and secondary structure levels, close homology is evident between the human and rabbit intestinal Na+/glucose cotransporters, and a significant homology is found between these and the Escherichia coli Na+/proline cotransporter (putP). No homology is detectible with other known proteins. We infer from these results that the mammalian Na+/glucose and prokaryote Na+/proline cotransporters share a common ancestral gene.

Amino Acid Sequence↗
PubMed1987

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl alpha-D-[14C]glucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (Vmax) of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/glucose cotransporter was found in a single fraction of approximately 2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.

Animals↗
PubMed1986

High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus.

An apparatus designed for preparative gel electrophoretic separation of proteins (M. A. Hediger, (1984) Anal. Biochem. 142, 445-454) has been used successfully for separating DNA restriction fragments. The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices. The amounts of DNA applied to the column range from a few micrograms to milligram quantities. Restriction DNA fragments very similar in size were isolated in pure form with the apparatus. After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing. Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA. The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h. In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.

DNA↗
PubMed1986

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, IV. The primary structure of the mesophilic lactate dehydrogenase from Bacillus subtilis.

The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B. X1) was determined. Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6). Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg). The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation. Lactate dehydrogenase from B. subtilis shows a 69% sequence homology to that from the thermophilic strain B. stearothermophilus, and a 34% sequence homology to those from higher organism. The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites). The relatively high sequence conservation of the lactate dehydrogenases from B. subtilis and B. stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.

Amino Acid Sequence↗
PubMed1985

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase.

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.

Amino Acid Sequence↗
PubMed1985

The amino acid sequence of thiogalactoside transacetylase of Escherichia coli.

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.

Acetyltransferases↗
PubMed1985

DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.

The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.

Acetyltransferases↗
PubMed1984

Apparatus and method for preparative gel electrophoresis.

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal alpha-amino groups is also described.

Amino Acid Sequence↗
PubMed1983

Isolation and partial characterization of the NH2-terminal membrane-binding domain of NADH-cytochrome b5 reductase.

The amphipathic flavoprotein NADH-cytochrome b5 reductase from steer liver was converted into the membrane-binding and soluble catalytic domains by controlled subtilisin proteolysis of vesicle-bound, externally oriented reductase. Amino acid analysis of the single nonpolar peptide which remained associated with the vesicles showed that it has a molecular weight of about 6400-6500 and that 65-66% of the amino acid residues are hydrophobic. Both the intact reductase and the nonpolar peptide were blocked to sequencing. Carboxypeptidase Y digestion of the holoenzyme and of a chymotryptically generated flavopeptide, which had lost membrane-binding capacity, released 1 residue of alanine and 2 residues of phenylalanine, indicating a common carboxyl terminus. Partial sequence analysis of the reductase placed the nonpolar peptide to the NH2-terminal side of the subtilisin-generated flavopeptide domain. Thus, the membrane-binding domain of cytochrome b5 reductase is localized at the NH2-terminus of the whole protein. The nonpolar peptide bound to vesicles contains a single fluorescent tryptophan with an emission maximum characteristic of a hydrophobic environment.

Amino Acid Sequence↗
PubMedDate not supplied

Expression cloning and cDNA sequencing of the Na+/glucose co-transporter.

Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.

Amino Acid Sequence↗