Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.

Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.     

31P-n.m.r. studies on cerebral energy metabolism under conditions of hypoglycaemia and hypoxia in vitro.

A system has been developed for performing 31P-n.m.r. studies on cerebral tissues superfused in vitro, and gives results comparable with those reported from studies in vivo. Under optimal superfusion conditions [10 mM-glucose and O2/CO2 (19:1)] the tissue concentrations of phosphocreatine and ATP were calculated to be approx. 3.1 and 1.3 mumol/g respectively. When the glucose of the superfusing medium was lowered to 0.5 mM, slightly decreased sugar phosphate peaks were observed, but there was no detectable change in [ATP] or [phosphocreatine]. At 0.2 mM-glucose, significantly decreased concentrations of phosphocreatine and ATP were observed. Substitution of pyruvate plus malate for glucose did not decrease levels of phosphocreatine and ATP. When the superfusing medium was gassed with air/CO2 (19:1; 'mild hypoxia'), there was an appreciable fall in sugar phosphates and phosphocreatine with no detectable effect on ATP. In the presence of N2/CO2 (19:1; 'severe hypoxia', since O2 was not completely excluded), concentrations of phosphocreatine fell considerably, but with little effect on ATP. The results demonstrate the feasibility of studying cerebral energy metabolism in vitro using the non-invasive 31P-n.m.r. technique and are discussed in relation to the sensitivity of cerebral tissues to metabolic insults in vitro and in vivo.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.     

Regiospecificity and stereospecificity in the enzymatic conjugation of glutathione with (±)-benzo(a)pyrene 4,5-oxide

¹³C-NMR analysis of the glutathione conjugates formed from (±)-benzo(a)-pyrene 4,5-oxide-4,5-¹³C by a purified glytathione transferase from little skate ($\text{Raja erinacea}$) liver demonstrated that equivalent amounts of the positional isomers (4,5-dihydro-4-hydroxy-5-glutathionylbenzo(a)pyrene and 4,5-dihydro-4-glutathionyl-5-hydroxybenzo(a)pyrene) were formed. Separation of these conjugates by HPLC and subsequent ¹³C-NME studies showed that only one diastereoisomer of each positional isomer was formed by the skate enzyme, each enantiomer of the arene oxide having produced only one of the two possible positional isomers. The non-enzymic reaction of (±)-benzo(a)pyrene 4,5-oxide with glutathione produced the four possible stereoisomers resulting from $\text{trans}$ addition to the epoxide ring. This was also true when rat liver cytosol was used as the source of transferase activity. The data demonstrate that skate liver glutathione transferase 4 has high substrate regiospecificity and stereospecificity for (±)-benzo(a)pyrene 4,5-oxide.     

Postreplication repair in Saccharomyces cerevisiae.

Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light (2 to 4 J/m2). Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were "chased," the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, we concluded that postreplication repair in excision-defective mutants (or leaky mutants) does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.     

Population Increase of Pratylenchus hexincisus on Corn as Related to Soil Temperature and Type

Population increase of Pratylenchus hexincisus on corn was tested over 3 months at 15, 20, 25, and 30 C in Marshall silt loam, Clarion silt loam, Buckner coarse sand, and Haig silty clay loam soils. The optimum temperature for increase was 30 C in all soils. The nematode population was significantly larger in Buckner coarse sand than in other soil types at 50 C. The recovered P. hexincisus populations equaled or exceeded initial inoculum levels at the two higher temperatures in Marshall silt loam and Haig silty clay loam and at 30 C in Clarion silt loam and Buckner coarse sand. P. hexincisus required 32,400 heat units in Haig silty clay loam and more than 40,000 heat units in the three other soil types to reach a level that is known to cause significant height and biomass reduction in corn under controlled condition.     

Effect of Ca ion concentration on cross-bridge kinetics in rabbit psoas fibers. Evidence for the presence of two Ca-activated states of thin filament

The effect of Ca ion concentration on cross-bridge kinetics in a small bundle (one to three fibers) of chemically skinned rabbit psoas muscle is studied. The length of the muscle is oscillated in small amplitude sine waves (0.2% L0 peak-to-peak) at varying frequencies (0.125 -- 167 Hz), and the resulting amplitude and phase shift in tension are measured. The frequency response function (complex stiffness) thus obtained can be divided into three parts, which we name process (A) (centered at 1 Hz), process (B) (3--17 Hz), and process (C) (50 Hz). Process (B), which represents oscillatory work, further splits into two processes (B' and B) at partial Ca activation (less than 50% P0), where the phase-frequency plot appears W-shaped. The slower of the two processes (B') disappears by full activation, at which time the plot appears V-shaped. The characteristic frequencies associated with the minima of the plot do not shift in a graded way with Ca concentration, indicating that there is no change in apparent rate constants. Apparent rate constants of processes (A) and (C) are minimally affected by Ca. The above results are not altered when ionic strength is changed between 128 and 265 mM. We propose that activated thin filaments can have two "on" states and that Ca concentration controls the distribution of these two states. This mechanism generally supports the "switch" hypothesis of Ca regulation.