Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.     

Energy-induced modulation of the kinetics of oxidative phosphorylation and reverse electron transfer

The kinetics of ATP synthesis by bovine heart submitochondrial particles (SMP) are modulated by the rate of energy production by the respiratory chain between two fixed limits characterized by apparent KmADP = 2-4 microM and Vmax approximately 200 nmol of ATP min-1 (mg of SMP protein)-1 at low energy levels and apparent KmADP = 120-160 microM and Vmax = 11,000 nmol of ATP min-1 (mg of SMP protein)-1 at high energy levels. These data indicate that KmADP and Vmax increase approximately 50-fold each; therefore, there is essentially no change in the catalytic efficiency of the ATP synthase complex in going from one extreme to the other. At intermediate rates of energy production, the kinetic data required introduction of a third, intermediate KmADP. A KmADP of 10-15 microM fitted all the data reported here and previously [Matsuno-Yagi, A., & Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038]. However, this is not meant to suggest that there is a fixed intermediate KmADP, as the transition from one fixed limit to the other may be fluid or involve more than one intermediate state. In addition, it has been shown that kinetic plots of SMP-catalyzed and ATP-driven reverse electron transfer from succinate to NAD are curvilinear and resolvable into a minimum of two apparent KmNAD values of about 20-30 and 200-300 microM. These results have been discussed in relation to the three potentially active catalytic sites of F1-ATPase and the structure of the NADH:ubiquinone oxidoreductase complex, the curvilinear kinetics of ATP hydrolysis, and changes in KmADP and KmPi in photophosphorylation as affected by the duration and intensity of light.     

Peroxidase activity in human red cell: a biological model for excited state molecules generation.

The presence of enzymically generated triplet acetone in red cells and energy transfer to eosin, rose bengal and 9,10-dibromoanthracene-2-sulfonate was indicate by: (1) product distribution; (2) K_ET τ_o, similar to the 2-methylpropanal/peroxidase/O₂ system; (3) correlation between hemolysis, oxygen uptake and photon emission; (4) membrane protection by energy acceptors, and (5) by comparison of the 2-methylpropanal/peroxidase/O₂ system with 2-methylpropanal/red cells/membranes/O₂ and 2-methylpropanal/acid extractable protein from red cells membrane/O₂ systems, which have a high peroxidase activity.This is the first report of a biological system producing a photohemolysis effect in the dark.     

Use of acyclovir in the prevention of herpes infections after allogenic bone marrow grafts

In a double-bind controlled study, oral Acyclovir has been compared to a placebo in a series of 39 consecutive patients undergoing bone marrow transplantation. A dose of 200 mg was given every 6 h from day 8 to day 35 after transplantation. Pharmacokinetic studies have shown the good absorption of the drug despite intestinal damage related to chemoradiotherapy or gut graft-versus-host disease (GVHD), there was no sign of toxicity. The protection against herpes simplex virus (HSV) infection was complete in the treated group when compared to the control group even in patients with high anti-HSV antibody titres. The same protection was observed against cytomegalovirus (CMV) infection. The incidence of HSV and CMV was the same in both groups after treatment ended. This study confirms the efficacy of Acyclovir against HSV infection and possibly against CMV infection when it is given prophylactically after bone marrow transplantation.     

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.     

Polyacetylenes in Hawaiian bidens

Leaves and roots of 19 species and six subspecies of Hawaiian Bidens were examined for polyacetylenes. Eleven C₁₃ hydrocarbons, aromatic and thiophenyl derivatives, one C₁₄ tetrahydropyran and three C₁₇ hydrocarbons were isolated all identified. All can be derived from a common precursor, oleic acid. Polyacetylenes were not detected in the leaves of 13 taxa although they are found in the roots of all. The occurrence of 2-[2-phenyl-ethyne-1-yl]-5 acetoxymethyl thiopene in Bidens has not been previously reported. Its ubiquitous presence is consistent with other evidence that the Hawaiian species are all derived from a single ancestral immigrant to the islands. Most taxa could be distinguished by their complement of polyacetylenes in roots and leaves. No variation was found to occur within taxa except in B. torta, in which each population had a unique array of polyacetylenes. Above the species level there appeared to be no taxonomically significant pattern to the distribution of polyacetylenes in this group.