Biomedical subjects

A W Abdulwajid

Publications and source records attributed to A W Abdulwajid.

3 recordsLinked to original sources

PubMed1986

Chemical modification of Escherichia coli RNA polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity and intrinsic zinc binding.

RNA polymerase (RPase) from Escherichia coli contains five subunits (alpha 2 beta beta' sigma) and two intrinsic Zn ions located in the beta and beta' subunits. This enzyme was rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The difference spectrum of the DEP-inactivated and native RPases showed a single peak at 240 nm indicating the formation of N-carbethoxyhistidines. No decrease in absorbance at 278 nm, due to O-carbethoxytyrosine, or modification of amino and sulfhydryl groups was observed. Inactivated RPase with six to nine histidines being modified could be fully reactivated by incubation with 0.5 M hydroxylamine at pH 6.0 and room temperature for 1 h. No structural difference was detected between the native and modified enzymes as evidenced by UV/visible and fluorescence spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern, or gel filtration properties. Substrate ATP at 0.11 and 1.14 mM concentrations provided, respectively, 25% and 90% protection against DEP inactivation, while template DNA did not. These results suggest that one or more histidine residues is/are in close proximity to the substrate binding site. The pH dependence of the DEP inactivation of RPase suggested the modification of histidine at the active site with a pK value of 6.9. The inactivation of RPase by DEP and the formation of N-carbethoxyhistidine displayed a similar second-order rate constant of approximately 0.9 mM-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-Directed RNA Polymerases↗

PubMed1986

Synthesis and properties of adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate: a fluorescent nucleotide substrate for DNA-dependent RNA polymerase from Escherichia coli.

A new fluorescent ATP analog, adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate (gamma-1,5-EDANS)ATP, containing the fluorophore N-(aminoethyl)-5-naphthylamine-1-sulfonic acid attached via a gamma-phosphoamidate bond was synthesized in good yield. It has absorption maxima at 255 and 344 nm and a fluorescence emission maximum at 490 nm. These spectral characteristics permit its uses as an energy acceptor for energy transfer from the intrinsic protein fluorophores and as an energy donor for the energy transfer to the intrinsic Co of Co-substituted RNA polymerases. This analog is a good substrate for Escherichia coli RNA polymerase and can be used to initiate the RNA chain. Incorporation of this analog into the total RNA synthesized was about 60% of that observed for ATP, independent of the templates used. Its Km values (22 and 118 microM) are twofold higher and its Vmax values (45 and 59 nmol/min/mg of enzyme) are 40% lower than those for ATP using calf thymus DNA and poly[d(A-T)], respectively, as template. For abortive initiation reaction using pAR1435 plasmid DNA as template, the Km and Vmax values of this analog are 2.7 times higher and 7 times lower, respectively, than those of ATP. With its desirable spectroscopic properties, (gamma-1,5-EDANS)ATP is a good probe for the studies of nucleotide-protein interactions, active site mapping of RNA polymerase, and other ATP-utilizing biological systems.

Adenosine Triphosphate↗

PubMed1983

Nickel-sequestering renal glycoprotein.

Kidney is the target organ where nickel is accumulated and subsequently excreted in the urine after an intraperitoneal administration of 63NiCl2 in rats. The radioactive nickel is found mostly bound to a low molecular weight protein in kidney, which was isolated, purified, and partially characterized in this study. Homogeneity of this protein was determined by polyacrylamide gel electrophoresis. Amino acid analysis showed the presence of high amounts of glycine and proline and low amounts of phenylalanine, tyrosine, hydroxyproline, and hydroxylysine. The protein was found to be a glycoprotein with a carbohydrate content of 10% (wt/wt). Preliminary carbohydrate analysis showed that this glycoprotein is a high mannose-type containing mannose, galactose/glucose, and glucosamine. On the basis of the amino acid and carbohydrate analyses, the molecular weight of the glycoprotein is about 15,000-16,000. In vitro addition of nickel to the kidney cytosol also showed the presence of this protein. The protein appeared not to be affected or altered in its nickel-binding capacity by intraperitoneal or intravenous administration of actinomycin D, indicating it to be a noninducible protein. The glycoprotein demonstrated many characteristics of renal basement membrane. It is proposed that this protein is either a part of the renal basement membrane or is a part of the procollagen in the process of its conversion to collagen of the renal basement protein. The protein has a high affinity for nickel. It also may possess a similar binding affinity for other metals as well and may constitute a natural process of handling toxic levels of metals to be excreted.

Amino Acids↗