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S L Mowbray

Publications and source records attributed to S L Mowbray.

52 records · Page 3Linked to original sources
PubMed1985

Purification and characterization of the aspartate chemoreceptor.

The chemoreceptor for aspartate in Salmonella typhimurium was purified from an Escherichia coli strain containing a plasmid bearing the receptor's structural gene (tar). The receptor was solubilized from salt-washed membranes with the nonionic detergent octyl-beta-D-glucopyranoside and purified by a combination of ion exchange, molecular sieve and hydroxyapatite-agarose chromatography. The inclusion of glycerol and 1,10-phenanthroline in all buffers used prior to ion exchange chromatography prevented scission of the receptor by an endogenous proteolytic activity. The solubilized receptor was estimated to have a molecular weight of 248,000 from its behavior on Sephacryl S-300, suggesting that the receptor may be organized as a multimer containing 4 +/- 1 identical subunits. Circular dichroic measurements of the purified protein indicate that 78% of its residues are arranged in helical secondary structures.

Bacterial Proteins↗
PubMed1985

Proteolytic fragments identified with domains of the aspartate chemoreceptor.

Two proteolytic fragments generated during the preparation of the aspartate receptor from Salmonella typhimurium have been purified. These fragments are the products of a single cleavage by an endogenous protease after amino acid 259 in the sequence of the intact receptor. Proteolytic fragment 1 (PF1) represents amino acids 1-259 (Mr = 29,000); this unit retains the aspartate-binding function of the intact receptor. The second fragment (PF2) includes residues 260-552 (Mr = 31,000) and has the normal sites of reversible methylation for the receptor. Like the purified intact receptor, this fragment can be methylated in vitro, although at a much slower rate. Circular dichroic measurements suggest that both proteolytic fragments contain substantial alpha-helical structure, approximately 95 and 53% for PF1 and PF2, respectively. No beta-structure could be detected in either fragment. Molecular sieve chromatography in the presence of detergent suggests that PF1 occurs as a stable multimer of an order equivalent to that observed for the detergent-solubilized aspartate receptor, i.e. a tetramer (+/- 1). PF2 is found to have a multimeric form which is sensitive to the removal of detergent. It is proposed that these fragments represent structural and functional domains of the aspartate receptor.

Amino Acid Sequence↗
PubMed1985

Neodymium:YAG laser interaction with intraocular lenses: an in vitro toxicity assay.

Use of the Nd:YAG laser is an effective technique to open an opacified posterior lens capsule. However, in the presence of a posterior chamber intraocular lens (IOL), precise focusing of the laser on the capsule is required to avoid pitting the lens optic. The question has been raised whether toxic products may result from laser damage to the IOL. We addressed this issue in the present study by exposing primary human corneal endothelial cell and human corneal organ cultures to solutions produced by purposefully hitting IOLs immersed in cell growth medium with a Nd:YAG laser. The lenses studied were lathe-cut polymethylmethacrylate (PMMA), injection-molded non-UV PMMA, injection-molded UV PMMA, and cast-molded UV PMMA. Samples of each material were irradiated in a holder containing 1 ml of cell culture medium using the following conditions: 5, 10, and 50 laser bursts at 10 millijoules (mJ), and 50 laser bursts at 5 mJ. The solutions were applied to the endothelial cell cultures (all materials) and to the corneal organ cultures (injection-molded non-UV lenses only). There was no toxicity in either assay for any of the materials studied.

Cells, Cultured↗
PubMed1983

The x-ray structure of the periplasmic galactose binding protein from Salmonella typhimurium at 3.0-A resolution.

The x-ray structure of the periplasmic galactose binding protein from Salmonella typhimurium, the specific receptor for taxis toward, and high-affinity transport of, galactose has been solved at 3.0-A resolution using multiple isomorphous replacement. The path of the polypeptide chain has been traced, and a model structure consisting of 292 amino acids has been fit to the electron density map. The overall shape of the molecule is that of a prolate ellipsoid, with dimensions 35 X 35 X 65 A. The protein consists of two similar domains of roughly equal size, related by an axis of pseudosymmetry, and separated by a deep cleft about 8 A wide. Each domain has a core of parallel beta sheet surrounded by five alpha helices, built by alternating strands of sheet and helix in a repeating pattern. Approximately 36% of the residues are involved in alpha helices, and 27% in beta sheet. The tertiary structure has been compared to that of the Escherichia coli arabinose binding protein (Gilliland, G.L., and Quiocho, F. A. (1981) J. Mol. Biol. 146, 341-362), a periplasmic receptor which is involved in transport, but not in chemotaxis. The overall folding of these two molecules is very similar, with the exception of two areas on the surface of the molecule on the long sides of the prolate ellipsoid. The observed variations are adequate to explain the differences in interaction of L-arabinose binding protein and galactose binding protein with the membrane proteins for transport and chemotaxis.

Calcium-Binding Proteins↗
PubMed1983

The introduction of specific sites for heavy metal binding in a crystalline protein.

Heavy metal derivatives of the galactose binding protein of Salmonella typhimurium were obtained by the treatment of crystals with carbon disulfide under anaerobic conditions, followed by exposure to mercury-containing reagents. Carbon disulfide reacts with protein amino groups to give a metastable dithiocarbamate, which is susceptible to covalent derivatization by mercurials. The number of amino groups which react for any particular crystalline protein will depend on the pH, the composition of the crystal mother liquor, and the steric accessibility limitations imposed by crystal packing. Direct reaction with protein crystals, rather than solution derivatization followed by purification and subsequent crystallization, is used to promote isomorphism of the derivative crystal with the native and to limit the number of available sites. For the S. typhimurium galactose binding protein, carbon disulfide treatment, followed by reaction with 2-chloromercuri-4-nitrophenol, resulted in binding at two sites at pH 8.0. Similar treatment with dimercury acetate gave one binding site for the dimercurial at the same pH. Both derivatives were isomorphous with the native crystal to a resolution of at least 3.5 A. These heavy atom derivatives have been used to produce an interpretable electron density map of the protein at 3-A resolution.

Calcium-Binding Proteins↗
PubMed1983

Estimation of the useful lifetime of polypropylene fiber in the anterior chamber.

A study of the stability of polypropylene fiber, size 4-0, to ultraviolet light was performed. Samples were radiated in physiological saline solution at 35.5 +/- 1.5 degrees C using a xenon-arc lamp at an intensity of 650 milliwatts per square centimeter. The light was filtered to remove wavelengths below 300 nanometers and thus simulated transmittance of the cornea. Tensile strength, elongation and modulus, and the surface morphology of the fiber by scanning electron microscopy (SEM) were determined as a function of exposure time. The average ultraviolet (UV) light intensity which irradiates the eye was determined from available meteorological data and was used to calculate human exposure at various geographical locations. This information along with the light energy of the xenon-arc lamp was used to calculate the useful lifetime of polypropylene fiber in the anterior chamber. Using an average exposure of 2.2 hours per day for a 60-year-old implant recipient, the useful lifetime ranges from 53 years in Miami, Florida, to 75 years in Seattle, Washington. Polypropylene loops from lenses which had been implanted in human eyes for up to 4.8 years showed no molecular degradation as compared to control material. Polypropylene fiber is therefore safe for use in anterior chamber implants.

Anterior Chamber↗
PubMed1979

A compound representing the D-glycerate terminus of the methylglucose-containing polysaccharide of Mycobacterium smegmatis.

In order to study the structure of the methylglucose-containing polysaccharide (MGP) of Mycobacterium smegmatis by NMR spectroscopy, we have prepared the model compound O-alpha-D-glucopyranosyl-(1 leads to 2)-D-glyceric acid. This compound, which represents the aglycon-containing terminus of MGP, was made from leucorse [O-alpha-D-glucopyranosyl-(1 leads to 5)-D-fructopyranose] by successive treatment with sodium borohydride, lead tetraacetate, and hypobromite. The structure of O-alpha-D-glucopyranosy.-(1 leads to 2)-D-glyceric acid was confirmed by chemical and enzymic methods. 13C and 1H NMR spectra of this compound, together with spectra of several disaccharides, were obtained for future reference in the polysaccharide study. The nine resonances in the 13C spectrum were assigned by comparison with the spectrum of methyl alpha-D-glucopyranoside. Analysis of the 1H NMR spectrum showed that the two methylene protons on C-3 of the glycerate moiety were less equivalent in the sodium salt than in the acid. This may be attributable to hydrogen bonding between the carboxylate and the hydrogen atom of the glycerate 3-hydroxyl group.

Carbohydrate Conformation↗
PubMed1975

Regulation of RNA synthesis in Escherichia coli during a shift-up transition.

These experiments investigate two aspects of RNA synthesis in Escherichia coli ML30 during the transition from a relatively slow rate of growth to a more rapid one: (1) the number of growing RNA molecules per cell, and (2) the average time required for addition of a nucleotide onto a growing RNA chain. Cells were grown at 30 degrees C in a glucose-minimal salts medium and shifted-up by the addition of Casamino acids. Measurements were made of the rates of incorporation over short intervals (e.g. 5,8,12, and 16 s) of [3-H]guanine into the internal and 3'-terminal nucleotides of RNA. After correction for the specific activities of the intracellular GTP pools, and for the rate of [3-H]guanine accumulation at the 3'-terminus of non-growing RNA, the rates of chain elongation were calculated. It was found that cells growing at a rate of 0.9 generations/h contain approx. 4800 RNA molecules, growing at a rate of 28 nucleotides/s per chain. Cells growing exponentially at the postshift-up rate (1.2 generations/h) contain 7000 RNA molecules per unit equivalent cell mass, which are growing at a rate of 32 nucleotides/s per molecule. Three min after shift-up, cells contain the same number or slightly fewer (10%) growing RNA molecules than cells prior to shift-up, 4300, and these are being elongated at a rate of about 32 nucleotides per s. The results are consistent with the view that in the range of growth rates studied, the total rate of RNA synthesis is regulated through a limitation in the number of functioning RNA polymerase molecules, each working at a relatively constant, presumably maximal, average rate.

Cell Division↗
PubMed1974

Generation of specific repeated fragments of eukaryote DNA.

Calf-thymus DNA, hydrolyzed with a site-specific endonuclease from Haemophilus influenzae Rd, yields 12 discrete bands on polyacrylamide-agarose gels. These range in size from 7.5 x 10(4) to 2 x 10(6) daltons, and they represent about 5% of the total DNA with individual fragments comprising 0.1-1.5%. The various DNA segments are repeated between 1500 and 220,000 times per haploid genome. Whereas the wide range of reiteration frequencies suggests different origins for some of the fragments, the bias in fragment densities in CsCl and in Ag(+)-Cs(2)SO(4) toward those of known satellite DNAs suggests similar origins for some of them. Models for the possible origin of the DNA fragments can be grouped into three distinct, experimentally distinguishable, classes.

Animals↗
PubMedDate not supplied

Structure of the periplasmic glucose/galactose receptor of Salmonella typhimurium.

The X-ray structure of the periplasmic glucose/galactose receptor (binding protein) of Salmonella typhimurium has been solved by the method of multiple isomorphous replacement and refined to a resolution of 2.4 A with an R-factor of 15.8%. The protein consists of two highly-similar structural domains with a bound sugar molecule trapped between. A combined sequence/structural approach is used to compare the structure of this protein to a similar arabinose receptor. The new sequence alignment obtained is used to analyze a number of features conserved for structural and functional reasons. This information allowed the prediction of similar features within the sequence of the analogous ribose receptor. Some of the consequences of the structures for chemotaxis and transport are discussed.

Amino Acid Sequence↗