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1.
J Wibbenmeyer L Brundage S R Padgette J J Likos G M Kishore 《Biochemical and biophysical research communications》1988,153(2):760-766
In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange. 相似文献
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C Crone J Frokjaer-Jensen JJ Friedman O Christensen 《The Journal of general physiology》1978,71(2):195-220
8.
Chemistry of the inactivation of cytosolic aspartate aminotransferase by serine O-sulfate 总被引:1,自引:0,他引:1
The reaction of serine O-sulfate with cytosolic aspartate aminotransferase [John, R.A., & Fasella, P. (1969) Biochemistry 8, 4477] has been reinvestigated. As in the corresponding reaction with beta-chloroalanine [Morino, Y., Osman, A.M., & Okamoto, M. (1974) J. Biol. Chem. 249, 6684], the enzyme is inactivated over a 10-min period, and the absorption maximum at pH 5.4 shifts from 430 to 336 nm. Upon prolonged standing the peak shifts again over a period of 20 h to 455 nm, a behavior entirely similar to that reported by Morino et al. for beta-chloroalanine in the presence of 3 M formate. When the pH of either the 10-min product (1a) or the 20-h product (1b) is raised to 11 or above, a yellow, diffusible compound (2) is released from the protein. This compound as well as its dephosphorylation and reduction products has been isolated and studied by NMR spectroscopy. Compound 2 is identical with a compound formed from serine sulfate and glutamate decarboxylase by a similar reaction sequence [Likos, J.J., Ueno, H., Feldhaus, R.W., & Metzler, D.E. (1982) Biochemistry (preceding paper in this issue)] and is the product of an aldol condensation of pyruvate with pyridoxal phosphate. When the 20-h product 1b is reduced with sodium borohydride and then heated in a boiling water bath, a material identical with the reduction product of 2 is released. We propose that the 20-h product 1b consists of 2 bound to the enzyme. Pathways for the formation of the various compounds are proposed. These findings require a reevaluation of the mechanisms of action of many enzyme-activated inhibitors of pyridoxal phosphate dependent enzymes. 相似文献
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The interaction between two stiff parallel DNA molecules is discussed using linear Debye-Hückel screening theory with and without inclusion of the dielectric discontinuity at the DNA surface, taking into account the helical symmetry of DNA. The pair potential furthermore includes the amount and distribution of counterions adsorbed on the DNA surface. The interaction does not only depend on the interaxial separation of two DNA molecules, but also on their azimuthal orientation. The optimal mutual azimuthal angle is a function of the DNA-DNA interaxial separation, which leads to azimuthal frustrations in an aggregate. On the basis of the pair potential, the positional and orientational order in columnar B-DNA assemblies in solution is investigated. Phase diagrams are calculated using lattice sums supplemented with the entropic contributions of the counterions in solution. A variety of positionally and azimuthally ordered phases and bundling transitions is predicted, which strongly depend on the counterion adsorption patterns. 相似文献
10.
We rigorously evaluated gender differences in the measurement validity of salivary testosterone. Matched serum, saliva, and finger stick blood spot specimens were collected from 40 (20 males) young adults (aged 18-27 years). Saliva was assayed for testosterone by two independent (isotopic and non-isotopic) immunoassay methods. Serum was assayed by commercially available immunoassay kits for free and total testosterone. An immunoassay was developed for the measurement of testosterone in dried blood spots and is presented in detail so as to be reproducible from this report. Regardless of assay method, salivary testosterone levels are modestly correlated with serum levels for males but not necessarily for females. Blood spot assay results were highly correlated with serum total and free testosterone for both males and females. Substitution of saliva assay results for serum values substantially underestimates known testosterone-behavior associations, and this effect is much more pronounced for females than for males. The findings have important implications for the use and potential misuse of noninvasive measures of testosterone, and with respect to statistical power, the probability of observing significant testosterone-behavior relationships. 相似文献