Positional isotope exchange

The detection of intermediates in enzyme-catalyzed reactions can be accomplished by several techniques. For those intermediates which do not have easily observed electronic spectra, use can be made of isotope exchange phenomena if the chemistry of the reaction is appropriate. Recently, the technique of positional isotope exchange (intramolecular isotopic scrambling) has been used to study several reactions which have been thought to involve high-energy intermediates in their mechanisms. A review of some of these reactions and the limitations of the method are presented in this article.     

Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods

The UTP-dependent ATPase reaction and the glutamine-dependent overall reaction of Escherichia coli CTP synthetase have been studied by rapid quench and isotope partitioning kinetics. The effect of GTP, an allosteric effector, on the pre-steady-state kinetics of both reactions has also been examined. The time courses of the UTP-dependent ATPase reaction in the presence and absence of GTP are both characterized by a burst of acid-labile phosphate equivalent to 0.93 and 0.43 subunits, respectively. The time course of the glutamine-dependent reaction in the absence of GTP is also characterized by a burst of acid-labile phosphate corresponding to 0.8 subunit; however, in the presence of GTP, no burst was observed. These results along with positional isotope exchange experiments [von der Saal, W., Anderson, P. M., & Villafranca, J. J. (1985) J. Biol. Chem. 260, 14997] provide evidence that the mechanism of CTP formation involves phosphorylation of UTP followed by attack of NH3, and finally release of phosphate, producing CTP, ADP, and Pi. A kinetic model for the first stages of the enzymatic reaction was developed from the rapid quench data, and the internal equilibrium constant for the formation of the phosphorylated UTP intermediate was determined. The internal equilibrium constants for the UTP-dependent reaction in the presence and absence of GTP were found to be 1.1 and 18, respectively. By contrast, the internal equilibrium constant for the reaction in the presence of glutamine was 50. Thus, the presence of glutamine shifts the internal equilibrium constant to favor formation of the phosphorylated UTP intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma hemoglobin determination in beagle dogs. An adaptation of the method of Crosby and Furth.

The plasma hemoglobin concentration of beagle dogs was measured following an improved bleeding technic which minimized trauma of the red cells. The benzidine reaction (benzidine dihydrochloride) was used to measure the hemoglobin present. Plasma hemoglobin values of the first 2 ml of blood collected were statistically significantly higher (P less than 0.01) than values of the next 2-3 ml of blood. Plasma hemoglobin values of normal beagles were 0.5-2.5 mg/dl in minimally hemolyzed samples. Recovery rates of up to 92% of hemoglobin added to plasma were possible with this method.     

Characterization of highly purified dopamine beta-hydroxylase

A modified purification procedure has been developed for dopamine beta-hydroxylase isolated from bovine adrenal medulla. Catalase is included in the homogenization step starting with a suspension of either chromaffin granules or adrenal medulla tissue. With this precaution, the enzyme remains stable in the supernatant solution in preparation for the subsequent purification step involving concanavalin A-Sepharose chromatography. The homogeneous enzyme has a specific activity in the range of 60-70 mumol O2 consumed/min/mg. Using radiolabeled metal ion chelators, it was determined that several of the chelators remained tightly bound to the enzyme after removal of the copper leading to difficulties in establishing stoichiometry of enzyme-bound metal ions.     

Characterization and kinetic studies of deglycosylated dopamine beta-hydroxylase

Dopamine beta-hydroxylase (D beta H) (EC 1.14.17.1) from adrenal medulla is a glycoprotein with approximately 5% carbohydrate by weight. The oligosaccharide chains of this enzyme were enzymatically removed with various glycosidic enzymes (endoglycosidases D, F, and H; glycopeptidase F; alpha-mannosidase; neuraminidase; and beta-galactosidase). The time course of deglycosylation was monitored by polyacrylamide gel electrophoresis, and evidence for sugar removal was shown by a modification of the Western blot technique utilizing 125I-labeled concanavalin A and by amino acid analysis. Protein was detected in the gel by using specific antibodies and 125I-labeled protein A. Steady-state kinetic data of deglycosylated D beta H show minor differences between the native and the deglycosylated protein. The Km values for tyramine were 2.17 and 1.66 mM whereas the Km values for oxygen were 0.18 and 0.14 mM for the native and the deglycosylated protein, respectively. The Vmax values (pH 5.0) for the two forms of the enzyme were comparable, with the deglycosylated D beta H being 15% lower. These data indicate that the oligosaccharide moieties present on D beta H do not play a role in catalysis.     

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.