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I Zabin

Publications and source records attributed to I Zabin.

At least 19 recordsLinked to original sources
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

Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12.

The sequences of the malE gene and of its mature product, the maltose-binding protein, have been determined and are in good agreement. The malE gene encodes the pre-protein (396 amino acid residues) which yields, upon cleavage of the NH2-terminal extension (26 amino acid residues), the mature maltose-binding protein (370 amino acid residues). The malE mRNA could form stable stem and loop structures, some of which may account for translational pauses observed by Randall et al. (Randall, L., Josefsson, L. G. & Hardy, S. J. S. (1980) Eur. J. Biochem. 107, 375-379). The sequence change due to an in-frame nonpolar deletion of 765 nucleotides in malE is also presented as well as homologies between the maltose-binding protein and other sugar-binding proteins.

ATP-Binding Cassette Transporters↗
PubMed1984

Purification of thiogalactoside transacetylase by affinity chromatography.

Thiogalactoside transacetylase, the product of the lacA gene of the lactose operon of Escherichia coli, has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration.

Acetyltransferases↗
PubMed1983

Purification, structure, and properties of hybrid beta-galactosidase proteins.

Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.

Amino Acid Sequence↗
PubMed1983

Nascent polypeptide chains exit the ribosome in the same relative position in both eucaryotes and procaryotes.

We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase. Similar studies with Escherichia coli ribosomes, using antibodies directed against the enzyme beta-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria. The eucaryotic ribosomal exit site is on the large subunit, approximately 75 A from the interface between subunits and nearly 160 A from the central protuberance, the presumed site for peptidyl transfer. This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined.

Escherichia coli↗
PubMed1982

beta-Galactosidase alpha-complementation. A model of protein-protein interaction.

Studies on beta-galactosidase alpha-complementation are reviewed. The isolation and structure of two beta-galactosidase fragments that form an enzymically active complex are described. One of these is a cyanogen bromide peptide from whole beta-galactosidase; the other is a dimeric protein from a lacZ deletion mutant of Escherichia coli. The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme. The overall reaction is essentially irreversible. A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide. alpha-Complemented enzyme contains overlapping sequences. Proteolytic experiments were carried out to determine the origin of the functionally important segment. The effect on alpha-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated. The implications of these results for beta-galactosidase structure and for proteins in general are discussed.

Amino Acid Sequence↗
PubMed1982

Inactivation of beta-Galactosidase by iodination of tyrosine-253.

Beta-Galactosidase is rapidly inactivated by iodination catalyzed by lactoperoxidase but is not inactivated in the presence of the substrate analogue, isopropyl beta-D-thiogalactoside (IPTG). Enzyme activity is lost upon the incorporation of 1 mol of iodine per mol of monomer, without dissociation of the tetrameric structure. Tryptic digests of beta-galactosidase iodinated with 125I in the presence and absence of IPTG were separated by high-performance liquid chromatography and were compared. One fraction was found to be more highly labeled in the digest from the inactivated protein. After isolation of the peptide, amino acid analysis indicated it to be Asp-Tyr-Leu-Arg, residues 252-255. Thus, Tyr-253 is the most reactive tyrosine in beta-galactosidase. This suggests that the conformation of this region of the protein may be altered by binding of IPTG to make Tyr-253 less accessible to iodination. Alternatively, Tyr-253 could be an active-site residue.

Amino Acid Sequence↗
PubMed1982

Lipolysis induction in adipocytes by a protein from tumor cells.

Extracts of thymic lymphoma that are obtained from AKR mice and are kept in the cold for at least several days can induce lipolytic activity in rat adipocyte suspensions. Freshly prepared extracts have low activity but contain a low molecular weight material of less than 10,000 daltons that aggregates on standing in the cold and becomes active. Treatment of aged extracts with trypsin causes a loss in activity indicating that the active material is a protein. It has been obtained in partially purified form, is relatively heat stable, and is not a lipase. Activity was also demonstrated in AKRXDBA/2 lymphoma (induced by AKR SL3-3 virus) and in transplanted lymphomas from a Friend-virus-induced erythroleukemia cell line in DBA/2 mice, but was not detected in normal thymus, spleen, liver, or other tissues. The partially purified material produced a massive fat mobilization when injected into normal mice.

Adipose Tissue↗
PubMed1982

Sequence studies on the maltose-binding protein of Escherichia coli.

The amino terminal sequence of the maltose-binding protein as well as the leader region were reported earlier [2]. The amino acid composition of the protein indicated that the protein has no cysteine and very few methionine, arginine and histidine residues. In contrast, the residues of lysine, alanine and aspartic acid (or asparagine) account for about a third of the approximately 360 amino acid residues in the protein. Five cyanogen bromide peptides ranging in size from 10 to 154 residues have now been isolated by Sephadex G50 and G100 gel filtration. These five peptides account for the whole protein. Sequence determination of these fragments as well as of others has now been initiated.

ATP-Binding Cassette Transporters↗
PubMed1981

beta-Galactosidase alpha-complementation. Overlapping sequences.

Enzyme activity is restored to two defective beta-galactosidase molecules (M15 protein lacking amino acid residues 11-41 and M112 protein lacking residues 23-31) by incubation with CNBr2 (residues 3-92 of beta-galactosidase). M15 and M112 proteins (alpha-acceptors) are dimers. Complemented enzyme, like wild type, has a tetrameric structure. Cleavage of CNBr2 with glutamic acid-specific protease yielded a much smaller alp ha-donor (3-41 peptide) which was also effective in complementation, indicating that the M15 protein can supply all of the residues from 42-92 for the structure of complemented enzyme. Treatment of M112 protein/3-41 peptide complemented enzyme with trypsin under very mild conditions followed by examination of the products demonstrated that the alpha-donor pep]tide supplies the NH2-terminal segment of complemented enzyme. Similar trypsin treatment of M15 protein/CNBr2 indicated that in this complemented enzyme the polypeptide region beyond those residues missing in the alpha-acceptor can be provided by either the alpha-donor or the alpha-acceptor. Both M15 protein and M112 protein are more susceptible to mild tryptic proteolysis than complemented enzyme, indicating a more open structure. Several antipeptide antibodies that react with these two proteins do not react with beta-galactosidase. M112 protein, like M15 protein, can be activated by anti-beta-galactosidase but to a much higher level.

Amino Acid Sequence↗
PubMed1981

Position of the lacZX90 mutation and hybridization between complete and incomplete beta-galactosidase.

The position of the termination codon in lacZX90 was determined by isolation of a lac+ revertant. Lysine was found to replace tyrosine at position 1,012 of beta-galactosidase, indicating that X90 protein lacked the carboxyl-terminal 10 residues. A heat- and urea-sensitive hybrid enzyme was formed in vivo when supC, which supplies tyrosine to the position in the polypeptide corresponding to the nonsense codon, was used to suppress lacZX90. This result shows that suppression that adds back the original amino acid may not lead to the production of the wild-type enzyme if the latter is multimeric, because incomplete chains can be incorporated into the oligomer.

Amino Acid Sequence↗
PubMed1980

A signal sequence is not sufficient to lead beta-galactosidase out of the cytoplasm.

Escherichia coli strains have been constructed in which lacZ, the gene for the cytoplasmic enzyme beta-galactosidase, is fused to lamB, the gene for an outer membrane protein. One such strain produces a beta-galactosidase which remains cytoplasmic even though it possesses the complete signal sequence of the lamB protein precursor at the amino-terminal end.

Amino Acid Sequence↗
PubMed1980

Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli.

The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell by virtue of an amino-terminal signal sequence. Using DNA sequencing, we have determined the precise nature of mutations in the signal sequence which prevent the export of the maltose binding protein, causing it to accumulate in the cytoplasm in its precursor form. In most cases, the change of a single hydrophobic or uncharged amino acid to a charged amino acid within the signal sequence is sufficient to block the secretion process.

ATP-Binding Cassette Transporters↗
PubMed1980

Positions of early nonsense and deletion mutations in lacZ.

The positions of three Escherichia coli lacZ operator-proximal nonsense mutations and one deletion mutation have been determined. The nonsense mutations were suppressed with supF, resulting in the production of active beta-galactosidase by each strain. Amino acid sequencing identified the positions of the tyrosine residues inserted by supF, and thereby established that nonsense mutations lacZ2, lacZ2246, and lacZU131 are at sites corresponding to amino acids 23, 36, and 41 of beta-galactosidase, respectively. The deletion mutant, lacZM112, produced a dimeric beta-galactosidase protein missing amino acid residues 23 through 31 of the native enzyme.

Amino Acid Sequence↗
PubMed1979

A dimer--dimer binding region in beta-galactosidase.

alpha Complementation in beta-galactosidase is the restoration of enzyme activity by addition of the alpha donor CNBr2, from amino acid residues 3--92 of the polypeptide, to inactive M15 protein from the lacZ deletion mutant strain M15. M15 protein lacks residues 11--41 and is a dimer; the active complex, like native beta-galactosidase, is tetrameric [Langley, K. E., & Zabin, I. (1976) Biochemistry 15, 4866--4875]. A dimer--dimer binding region in beta-galactosidase has been identified by proteolytic and immunologic studies of alpha-complementation. Proteolytic experiments were carried out with trypsin. Treatment of native beta-galactosidase with trypsin, followed by reaction of the mixture with cyanogen bromide, yields intact CNBr2 as measured by its ability to complement M15 protein. Active CNBr2 is not obtained when urea-denatured beta-galactosidase is treated in the same way. Therefore the segment corresponding to CNBr2 is apparently buried within the folded protein. Immunologic experiments were carried out with antibodies against CNBr2, tryptic peptide T8 (residues 60--140), and CNBr3 (residues 93--187). Anti-CNBr2 and anti-T8 bind to M15 protein but not to beta-galactosidase, indicating that this area is exposed in the dimer. Anti CNBr2, but not anti-T8 or anti-CNBr3, inhibits the formation of alpha-complemented enzyme. These results indicate that an early part of the sequence, within the segment corresponding to CNBr2, is involved in dimer--dimer interaction.

Antibodies↗