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Biomedical subjects

K A Taylor

Publications and source records attributed to K A Taylor.

At least 73 records · Page 4Linked to original sources
PubMed1987

Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli.

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.

Bacteroides↗
PubMed1987

Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli.

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).

Autoradiography↗
PubMed1987

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12.

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.

Antibodies, Monoclonal↗
PubMed1986

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum.

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.

Animals↗
PubMed1986

Image reconstruction using electron micrographs of insect flight muscle. Use of thick transverse sections to supplement data from tilted thin longitudinal sections.

Three-dimensional reconstruction using electron micrographs of thin sections is a powerful technique for determining cross-bridge structure. Tilt restrictions in the electron microscope prevent data collection beyond tilt angles of 60 degrees, giving rise to a "missing cone" of transform data. We show here how much of this data can be obtained using micrographs of thick transverse sections, and the effect this data has on reconstructed images of the insect flight muscle MYAC layer. As a byproduct, the analysis showed that section thinning resulting from prolonged electron irradiation had occurred in the thin longitudinal section used for the previously published MYAC layer reconstruction (Taylor et al., 1984). Comparison of projection density maps calculated from the thin longitudinal section reconstruction and the thick section data show that the data within the missing cone that is not accessible by tilting sharpens the boundaries of the components, flattens the density profile across the thick filament, and enlarges the molecular envelope of the thin filament. We conclude that the reconstructed images of the MYAC layer provide a picture of the structural principles underlying the system but that transform data within the missing cone are necessary to describe accurately the envelopes and profiles of these structural elements.

Actin Cytoskeleton↗
PubMed1985

Crystallization of the Ca2+-ATPase of sarcoplasmic reticulum by calcium and lanthanide ions.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop in sarcoplasmic reticulum vesicles exposed to Ca2+ or lanthanide ions. The Ca2+- or lanthanide-induced crystals are presumed to represent the E1 conformation of the Ca2+-ATPase, and their crystal form is clearly different from the earlier described E2 crystals induced by Na3VO4 in the presence of ethylene glycol bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid (Taylor, K. A., Dux, L., and Martonosi, A. (1984) J. Mol. Biol. 174, 193-204). Analysis of the crystalline arrays by negative staining or freeze-fracture electron microscopy reveals obliquely oriented rows of particles corresponding to individual Ca2+-ATPase molecules. Computer analysis of the negatively stained lanthanide-induced crystalline Ca2+-ATPase arrays shows that the molecules are arranged in a P1 lattice. The pear-shaped profiles of Ca2+-ATPase molecules seen in projection in the density maps are similar to those seen in vanadate-induced crystals. The space group and unit cell dimensions of the E1 crystals are consistent with Ca2+-ATPase monomers as structural units, while the vanadate-induced E2 crystals form by lateral aggregation of chains of Ca2+-ATPase dimers. The transition between the E1 and E2 conformations may involve a shift in the monomer-oligomer equilibrium of the Ca2+-ATPase. The formation of E1 crystals by PrCl3 is promoted by inside negative membrane potential, presumably through stabilization of the E1 conformation of the enzyme. Cleavage of the Ca2+-ATPase by trypsin into two major fragments (A and B) did not interfere with the Ca2+- or the Pr3+-induced crystallization.

Adenosine Triphosphate↗
PubMed1984

A cold stage for the Philips EM300 electron microscope.

A cold stage has been constructed for the Philips EM300 (and EM301) electron microscopes for investigating the structure of frozen-hydrated biological specimens. The stage entails minimal alterations to the instrument and is capable of a resolution better than 10 A at the normal operating temperature of -120 degrees C. Frozen specimens can be readily exchanged without condensation or warming up, and maintained in the stage over periods of several hours without detectable deterioration.

Freezing↗
PubMed1984

Analysis of the three-dimensional structure of the urinary bladder epithelial cell membranes.

The three-dimensional structure of the luminal membrane of the mammalian urinary bladder has been determined using electron micrographs of tilted membranes preserved in neutralized potassium phosphotungstate stain. The density map consists of 12 stain-excluding regions, 57 A thick, that are organized as an inner ring of 6 and an outer ring of 6, plus a small stain-excluding feature directly on the sixfold axis. Only the luminal surface of the membrane can be observed suggesting that at the resolution of the reconstruction the cytoplasmic surface is smooth.

Animals↗
PubMed1983

Three-dimensional arrangement of the cell wall protein of Sulfolobus acidocaldarius.

The three-dimensional structure of the S-layer that surrounds the bacterium Sulfolobus acidocaldarius is described in detail. Pieces of the S-layer, which are two-dimensional crystals with p6 symmetry, have been studied by crystallographic analysis of electron micrographs of tilted specimens. In the density map, each asymmetric unit appears to consist of several domains connected by strong hinges. On the basis of the ragged appearance of the structure at torn edges, we now suggest that the single species of polypeptide is in a highly extended conformation, with much overlap between different molecules, and show how such molecules might be weaved together to produce the morphological domains. We show that the closed surface lattice of the intact cell wall contains 5-fold and 7-fold vertices and show how the subunit structure appears to be well suited to form 5-fold and 7-fold symmetric rings at these points in place of the 6-fold rings of the hexagonal lattice.

Bacterial Proteins↗
PubMed1982

Plasma prostaglandin levels in radiation-induced enteritis.

Increased prostaglandin synthesis has been implicated as a causative factor in the production of radiation induced enteritis. Seventeen patients selected to begin pelvic irradiation for treatment of gynecological cancer had plasma Prostaglandin E, Prostaglandin F, and 13, 14 dihydro 15 keto PGF2 alpha metabolite determined by radioimmunoassay, prior to initiation of radiotherapy, at weekly intervals during treatment and at six weeks following completion of radiotherapy. A total of 362 prostaglandin determinations were performed. Thirteen patients (76%) developed significant diarrhea consisting of three or more watery bowel movements per day. Nine patients (53%) had intermittent colicky pain and six patients (35%) had nausea and vomiting during treatment. Statistical evaluation revealed no significant elevation of plasma prostaglandins during radiotherapy.

Adult↗