Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.     

Absence of Detectable Mitochondrial Recombination in Paramecium

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

S-trifluoroacetonyl-coenzyme A:a 19F analogue of acetyl-coenzyme A

S-Trifluoroacetonyl-coenzyme A has been synthesized in 87% yield by reaction of 1,1,1-trifluoro-3-bromopropanone with trilithium coenzyme A in presence of pyridine. The compound was characterized by its ultraviolet absorption spectrum and 1H and 19F nuclear magnetic resonance spectra. The alpha-methylene protons of the S-trifluoroacetonyl group exchanged with D2O and showed a pKa of 9.85 in S-trifluoroacetonylmercaptoethanol. S-Trifluoroacetonyl-coenzyme A is a competitive inhibitor of porcine heart citrate synthetase (Ki = 0.16 mM). It forms a binary complex with the enzyme and a ternary complex with enzyme/oxaloaetate binary complex, as evidenced ty the 19F shift. S-Trifluoracetonyl-coenzyme A and S-trifluoroacetonylmercaptoethanol form weak to moderately strong complexes with alpha-cyclodextrin and show little or no interaction with the methylglucose polysaccharide and lipopolysaccharides from Mycobacterium smegmatis [Smith, W. L., & Ballou, C. E. (1973) J. Biol. Chem. 248, 7118]. S-Trifluoroacetonylmercaptoethanol probably forms an inclusion complex with alpha-cyclodextrin because the interaction is reversed by compounds that do form inclusion complexes.     

Chiroptical and stoichiometric evidence of a specific,primary dimerisation process in alginate gelation

The stoichiometry of calcium-ion chelation to alginate chains has been investigated by circular dichroism (c.d.), and by equilibrium dialysis in the presence of various concentrations of sodium chloride. C.d. intensity in the carboxylate π → π * spectral region increases linearly with calcium-ion concentration up to a level equivalent to half the total poly-L-guluronate stoichiometric requirement, and thereafter shows little further change. Similarly, the level of bound calcium resistant to displacement by swamping concentrations of sodium ions is equivalent to half the stoichiometric requirement of poly-L-guluronate chain-sequences alone. In terms of the previously developed “egg-box” model of co-operative junction-zone formation in alginate gelation, these results are interpreted as showing that the primary mechanism of interchain association is by dimerisation of poly-L-guluronate chain-segments in a regular, buckled, two-fold conformation related to that characterized for the free acid in the solid state, with tight interchain chelation of calcium to the carboxylate groups on the interior faces of the dimer (i.e., half the carboxylate residues of the participating chain-sequences). This interpretation is entirely consistent with previous evidence from electron microscopy, and offers a simple rationalisation of experimental results from competitive-ion binding studies.     

Myocardial Revascularization in High-Risk Coronary Patients

It is recognized that postoperative mortality, infarction and the need for inotropic support are increased following myocardial revascularization in highrisk patients. Operations were carried out in 57 such patients in whom one or more of the following factors were present: ventricular dysfunction—ejection fraction less than 0.4 (17), unstable (8) or preinfarction angina (29), evolving infarction (8), recent infarction (less than two weeks before) (5) and refractory ventricular tachyarrhythmia (4). Combined risk factors were present in nine patients. The following principles were utilized to minimize ischemic injury: (1) avoidance of prebypass hypertension and hypotension, (2) avoidance of extreme hemodilution, (3) avoidance of ventricular fibrillation, (4) maintenance of beating empty heart, when possible, (5) the limiting of ischemic periods to less than 12 minutes (hypothermia 32°C) and (6) repaying myocardial oxygen debt with total (vented) bypass, when necessary. The following results were obtained: inotropic support was required in five patients (9 percent), “new” postoperative infarction occurred in five patients (9 percent) and one patient died (2 percent). These results are comparable to those reported in good-risk patients, and indicate that optimal myocardial protection will allow safe revascularization in a high-risk patient.     

Characterization of a mannan-like oligosaccharide from Mycobacterium smegmatis

A nitrogen-free neutral mannooligosaccharide, similar in structure to the polysaccharide component of yeast mannoproteins, has been isolated from Mycobacterium smegmatis ATCC-356. It has a molecular weight of 3200 and is terminated at the reducing end by mannose. nuclear magentic resonance spectroscopy, methylation analysis, selective enzymic degradation and acetolysis indicates that the molecule consists of an alpha1 --> 6-linked backbone to which single mannose units are attached in alpha1 --> 2 linkage as sidechains.     

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation

Summary A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.