Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Rapid identification of microorganisms by circular-intensity differential scattering

There is a pressing need in clinical medicine for rapid identification of microorganisms. We describe a method that has the potential for such rapid identification: circular-intensity differential scattering, which is based on the differential scattering of left and right circularly polarized light. The scanning time required to obtain the spectral signature of an organism is about 4 min. Using a commercial circular dichrograph modified to measure circular intensity differential scattering at 90 degrees, we obtained significantly different spectra for five different crude influenza viruses. Salmonella typhimurium TA98 and TA100, and Escherichia coli HB101, HB101(pBR322), and HB101(pMB9). Purified supercoiled plasmid pBR322 DNA was readily distinguishable from linear pBR322 DNA; such differences in nucleic acid packaging may be significant factors in the discriminatory power of this technique.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.     

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Quantum yield and image contrast of bacteriochlorophyll monolayers in photoelectron microscopy.

The photoelectron quantum yield spectrum of bacteriochlorophyll aGg (Bchl a ) from Rhodospirillum rubrum was determined in order to evaluate the possibility of mapping photoreceptor distribution and organization in bacterial chromatophores. The quantum yield is on the order of 1 X 10(-3) electrons/incident photon at 180 nm and decreases to 2.5 X 10(-5) electrons/incident photon at 230 nm. Photoelectron micrographs confirm the high contrast predicted between monolayers of Bchl a against a lipid background (calcium arachidate). A significant contrast difference is found between the two monolayer orientations, demonstrating that photoelectron microscopy is a sensitive detector of asymmetry in Bch1 a monolayers.     

The effect of chromosomal polytenization on the rates of RNA synthesis and decay in the salivary glands of the blowfly, Calliphora erythrocephala

The rates of total RNA synthesis and accumulation have been measured in the polytenic salivary gland cells of the blowfly, Calliphora erythrocephala, by three methods: (1) injecting larvae with [2-³H]adenosine and determining its flow into the cellular ATP pool and RNA, (2) measuring the increase in glandular RNA optically, and (3) measuring the rate of flow of ATP out of the cellular pool. The size of the steady-state pool of rapidly turning over RNA and its half-life, were calculated from these kinetic data and, also, by an independent measurement of the steady-state content of nuclear RNA. These parameters were compared at a number of developmental stages which differed in degree of chromosomal polytenization. The results indicate that these polytenic cells synthesize RNA at a rate approximately 10³ times those of other diploid eukaryotic cells. This rate is independent of the increase in chromosomal polyteny that accompanies larval development. Approximately 67% of the newly synthesized salivary gland RNA is an unstable component with an average first-order half-life of 20–25 min. The remainder is a long-lived species with an estimated average first-order half-life of about 30 hr.     

Chromosome analysis on 930 consecutive newborn children using quinacrine fluorescent banding technique

Summary Chromosome analysing using quinacrine fluorescence was performed on 930 consecutive newborn infants. The total incidence of major chromosome aberrations including numerical changes of the sex chromosomes, and structural changes of autosomes, was 0.54%. Incidences of XYY (0.4%) and XXY (0.2%) were relatively higher as compared to other studies. About 0.75% of the newborn infants were found to have a variable bright fluorescent band located on the proximal area of the short arm (p11) rather than on the proximal long arm (q11) of chromosome No. 3. Attempts were also made to record the variable fluorescent regions on 7 autosomes and the Y chromosome.     

Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities.

Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes.