Relative activities of linear and cyclic electron flows during chloroplast CO2-fixation

Evolution of oxygen and turnover of cytochromes b-563 and ? were measured upon illumination of isolated intact spinach chloroplasts with a series of flashes. The flash yield of cytochrome ? oxidation approximated the sum of the yields of cytochrome b-563 reduction and electron transfer through Photosystem II, regardless of whether HCO^?₃, 3-phosphoglycerate or O₂ served as the terminal electron acceptor. No absorbance contribution from cytochrome b-559 was discerned within the time range studied. Some pseudocyclic electron flow occurred when both HCO^?₃ and 3-phosphoglycerate were omitted, and possibly also during induction of photosynthesis; however, the flash yield data suggest that O₂ is not reduced at a significant rate during steady state photosynthesis. The maximum rate of cytochrome ? turnover (1000 μequiv./mg chlorophyll per h) was adequate to support the highest rates of photosynthesis observed in isolated chloroplasts.These results agree with the concept that cytochrome ? is a component both of the linear and cyclic pathways whereas cytochrome b-563 functions only in the cyclic pathway. NH₄Cl decreased the half time of cytochrome b-563 oxidation from 11.6 to 8.2 ms and decreased the half time of cytochrome ? reduction from 7.2 to 2.8 ms. The cyclic and linear pathways thus seem to be jointly regulated by a transthylakoid H⁺ gradient through a common control point on the reducing side of cytochrome ?. Cyclic turnover also increased during the induction phase of photosynthesis, when linear throughput is limited by the rate of utilization of NADPH. The slow rise in the P-518 transient correlated with increased cyclic activity under the above conditions.It is proposed that flexibility in the utilization of linear and cyclic pathways allows the chloroplast to generate ATP and NADPH in ratios appropriate to varying needs.     

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor

Summary 80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR ⁺ strain but not by corresponding preparations from an argR ^- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on -galactosidase synthesis.     

Lanosterol 14α-demethylase. Similarity of the enzyme system from yeast and rat liver

The chemical synthesis of 24,25-dihydro[32-¹⁴C]lanosterol is described. The incubation of this material with a cell-free system from $\text{Saccharomvoes cerevisiae}$ or with a microsomal preparation from rat liver resulted in both cases in the release of [¹⁴C]formic acid. This result suggests that in the biosynthesis of ergosterol in yeast, as well as in that of cholesterol in higher animals, the 14α-methyl group of lanosterol is removed as formic acid. In both systems, the measurement of the rate of release of [¹⁴C]formic acid from 24,25-dihydro[32-¹⁴C]lanosterol provides a simple and direct assay of lanosterol 14α-demethylase. Carbon monoxide inhibited both yeast and liver 14α-demethylase.     

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes

Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M₄ lactate dehydrogenase structures. One of these is the 2.0 Å resolution apoenzyme structure; the other is a 3.0 Å resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase²: NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.     

Purification,properties, and mode of action of hemicellulase I produced by Ceratocystis paradoxa

A culture isolate (CP₂) of the fungal plant pathogen Ceratocystis paradoxa produces at least five extra-cellular hemicellulases when grown on a medium containing a commercial hemicellulose as inducer. One of the five enzymes, hemicellulase I (HC-I), was purified by ammonium sulphate precipitation, ion-exchange chromatography (DEAE-Sephadex and then Cellex-CM), and iso-electric focusing at pH 3–10 and 8–10. HC-I behaves as a single protein on electrophoresis at pH 6.0 and 8.4. The enzyme degrades hemicellulose B (an arabino-4-O-methylglucuronoxylan) and arabinoxylan to arabinose, xylose, xylobiose (Xyl₂; β-D-Xylp-(1→4)-D-Xyl), and a mixture of arabinose-xylose and xylose oligosaccharides (AraXyl_n and Xyl_n, where n  3, 4, or 5). The enzyme is deduced to be an endo-enzyme. Xylotetraose (Xyl₄) was the lowest homologue of the xylose oligosaccharides attacked, yielding xylobiose and xylotriose (Xyl₃) only. A mechanism is postulated for this reaction. AraXyl₂AraXyl₅ were slowly hydrolysed to arabinose and the respective xylose saccharide (Xyl₂Xyl₅), and thence to Xyl₂ and Xyl₃. Hydrolysis of the arabinofuranosyl linkage probably does not occur at the same active site as for the xylose oligosaccharides. Hemicellulose B fractions from different sources appeared to be degraded by HC-I. The enzyme showed optimum activity at pH 5.5 and 40°, and K_m was 4.24 mg of hemicellulose/ml.