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Biomedical subjects

A V Fowler

Publications and source records attributed to A V Fowler.

At least 37 records · Page 2Linked to original sources
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

The adenovirus hexon protein. The primary structure of the polypeptide and its correlation with the hexon gene.

The primary structure of the adenovirus hexon polypeptide has been determined by amino acid sequence studies of peptides from all regions of the molecule combined with sequence analysis of selected areas of its gene. The sequence presented contains 966 unique amino acid residues. Overlapping peptides recovered from CNBr cleavage and from digestions with proteolytic enzymes were analyzed, as well as DNA segments around sites for restriction endonucleases in the hexon gene. The primary structure is in good agreement with the total composition of the protein, with the compositions of individual CNBr fragments, and with known locations of restriction enzyme cleavage sites in the gene. Distinct regions of internal homology do not occur in the structure. The entire hexon polypeptide is encoded by a contiguous DNA sequence without intervening sequences.

Adenoviruses, Human↗
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

The araC gene of Escherichia coli: transcriptional and translational start-points and complete nucleotide sequence.

The nucleotide sequence of the Escherichia coli araC gene and flanking regions have been determined from a series of overlapping fragments using the technique of base-specific chemical cleavage of Maxam and Gilbert (1980). The nucleotide sequence of araC gene was confirmed by the partial amino acid sequences of araC protein and its methionine peptides. The primary structure of araC polypeptide consists of 291 amino acid residues, giving it a chemical molecular weight of 33 314 daltons. The transcriptional start-point has been deduced from the sequence of araC mRNA synthesized in vitro and in vivo, and it is located 148 bp away from the transcriptional start-point of the araBAD operon. The translational start-point for the araC gene was deduced from the N-terminal amino acid sequence of the protein, and is located 165 bp from the 5'-end of araC mRNA. There is, therefore, a leader sequence of 164 bp preceding the araC gene.

Arabinose↗
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

Effects of social and environmental change on institutionalized mentally retarded persons: the relocation syndrome reconsidered.

The "relocation syndrome" in multiply-handicapped, institutionalized mentally retarded residents was examined in an observational study. Weight change and the initiation and duration of constructive and nonconstructive behavior was observed, as well as resident-staff interactions before and after relocation to a new living unit. The hypothesis that there would be an increase in behavior deleterious to the health of clients after relocation, when compared to two premove baseline periods of crowding, was not totally supported. Although relocation did cause significant changes in a variety of behaviors, not all were deleterious (i.e., no mortalities, weight gains). Results were discussed in terms of a social-situational model.

Adolescent↗
PubMed1978

Probes of beta-galactosidase structure with antibodies. Reaction of anti-peptide antibodies against native enzyme.

Antibodies were prepared against 18 tryptic and cyanogen bromide peptides from beta-galactosidase ranging in size from 15 to 96 amino acid residues representing more than 80% of the polypeptide chain. They were tested for binding capacity and affinity toward their homologous antigens and toward the whole native protein. Nine antisera bound to beta-galactosidase; these had been raised against certain peptides from the central and carboxyl-terminal regions of the poly-peptide chain. Based on these results a preliminary model of the three-dimensional structure of the folded protein is suggested.

Antibodies↗
PubMed1978

Probe of beta-galactosidase structure with iodoacetate. Differential reactivity of thiol groups in wild-type and mutant forms of beta-galactosidase.

Carboxymethylation with 14 C-labeled iodoacetate of cysteine residues in wild-type beta-galactosidase from Escherichia coli and in a defective beta-galactosidase from deletion mutant strain M15 was investigated in order to determine accessible positions in the tetrameric wild-type form and the dimeric mutant M15 protein. The extent of carboxymethylation, the effects on biological activity, antibody activation, physical stability, and the labeling of particular residues were studied. The results distinguish three groups of spatial relationships for cysteine residues in the protein, define possible regions for subunit interactions, and confirm that no cysteine residue is specifically involved in catalysis. Residue 1019 and to a lesser extent 498 are accessible in the tetrameric protein and probably represent exposed areas. In the M15 protein, these two, and three additional residues, at 76,387 and 600, were found to react significantly with reagent. One or more of the latter are suggested to be in the dimer-dimer interface. Complementation and activation by antibody are inhibited by carboxymethylation of M15 protein.

Binding Sites, Antibody↗
PubMed1978

Amino acid sequence of beta-galactosidase. VI. Limited tryptic digestion of the citraconylated protein and sequences of tryptic peptides.

Hydrolysis with trypsin of citraconyl-carboxy-methyl-beta-galactosidase was carried out under limiting conditions. No Asp-Arg-X sequences were cleaved and many large peptides were produced. Butanol extraction from dilute acid proved very useful for separating the more hydrophobic fragments. Peptides were purified and sequenced. From this digest and two earlier preparations, all 80 theoretically possible tryptic fragments have been isolated and their structures determined.

Amino Acid Sequence↗
PubMed1978

Methionine 500, the site of covalent attachment of an active site-directed reagent of beta-galactosidase.

The site of attachment to beta-galactosidase of the active site-directed inhibitor, beta-D-galactopyranosylmethyl p-nitrophenyl triazene, was determined. When the enzyme is completely inactivated, 1 mol of the galactopyranosylmethyl group is bound per mol of monomer with retention of the tetrameric structure. After reaction with the [14C]methyl reagent, labeled peptides were isolated and analyzed. The radioactive label was found to be covalently bound to methionine residue 500.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of beta-galactosidase. V. Isolation and sequences of chymotryptic peptides.

The amino acid sequence of 72 chymotryptic peptides isolated from 14C-, 3H-labeled carboxymethyl-beta-galactosidase has been determined. A variety of techniques were used in the isolation procedures including separation by solubility, size, and ion exchange and paper chromatography. These peptides contain approximatley 500 amino acids, range in size from 2 to 26 residues, and give overlaps with tryptic peptides of 16 to 55 residues. Peptides from this digest and those reported earlier from tryptic digests account together for the sequence of about 600 of the 1021 residues in the subunit.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of beta-galactosidase. VII. Isolation of the 24 cyanogen bromide peptides.

All of the 24 cyanogen bromide peptides of beta-galactosidase have been isolated in pure form. Of these 8 ranged in size from 2 to 5 residues and were purified by paper electrophoresis. The 16 large peptides, from 23 to 119 residues, were chromatographed at pH 5.0 on a carboxymethyl-cellulose column in 0.02 M ammonium acetate buffer containing 8 M urea. A number of peptides were obtained in pure from following Sephadex G-50 or G-75 gel filtration. Others were separated on sulfopropyl-Sephadex or diethyl-(2-hydroxylpropylaminoethyl)-Sephadex. There large peptides were obtained in over 50% yield and several others were obtained in more than 25% yield.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of beta-galactosidase. VIII. Sequence of the NH2-terminal segment, CNBr peptides 1 to 9, residues 1 to 377.

The amino acids in 9 cyanogen bromide peptides have been placed in sequence starting from the NH2 terminus. The peptides account for residues 1 to 377 of the whole protein and include the largest (CNBr7, 119 residues) and the smallest (CNBr1, 2 residues) of the cyanogen bromide peptides. This region contains only 3 of the 20 lysine residues in the polypeptide chain. A high proportion of charged groups are present (28 of 66 arginine, 28 of 60 glutamic acid, and 24 of 65 aspartic acid residues).

Amino Acid Sequence↗