ALBERT

Notes: Summary The incorporation of [35S]-sulphate was followed into washed-cell suspensions of Nitrobacter agilis. Thus, bound sulphate, sulphite, sulphide, cysteine, glutathione, homocysteine, methionine and taurine were detected in the ethanol-soluble fraction as well as in the residual hydrolysed fraction. The reaction between thiol groups and N-ethylmaleimide has been successfully used to stabilize the SH-compounds in cell extracts, and the derivatives thus obtained were separated by paper chromatography. A soluble enzyme system catalyzing the reduction of sulphate to sulphite has been prepared. As a result of DEAE-cellulose-11 column chromatography, the enzyme complex was cleaved into two protein bands, one containing ATP-sulphurylase and the other APS-kinase and PAPS-reductase. The last two enzymes were further purified by DEAE-sephadex and Sephadex G-150 column chromatography. At pH 7.6 the enzymes show maximal activity in the presence of ATP and an ATP-generating system (creatine phosphate and creatine phosphokinase), APS, NADP+, a NADP+-reducing system (glucose-6-phosphate and a glucose-6-phosphate dehydrogenase) and MgCl2. Addition of small amounts of 2,3-dimercaptopropan-1-ol (BAL) to the buffers stabilized the enzymes and enabled them to be dialyzed for 16 h, without loss of activity. Anaerobic conditions are required for maximal activity. The optimum concentration of various cofactors for enzyme activity has been determined. The K m values are as follows: ATP, 1.3×10-3 M; APS, 1.6×10-4 M and NADP+, 1.8×10-3 M. The molecular weight of the APS-kinase and PAPS-reductase complex is about 280000. The PCMB inhibition of the two enzymes is reversed by adding GSH, L-cysteine and Cleland's reagent.

Notes: Summary The incorporation of [35S]sulphate was followed into the washed cell suspensions of Nitrosomonas europaea. Thus bound sulphate, sulphite, sulphide, cysteine, glutathione, homocysteine and methionine were found in the ethanol soluble fraction as well as in the residual hydrolysed protein fraction. Cysteic acid, methionine sulphoxide and methionine sulphone were detected in the residual protein. The reaction between sulphydryl groups and N-ethylmaleimide has been successfully used to stabilize the thiol compounds in cell-extracts and the derivatives thus obtained were separated by paper chromatography. As in other microorganisms, sulphate is first activated by ATP in Nitrosomonas before it is reduced. The formation of APS and PAPS has been studied. A pathway for the incorporation of [35S]sulphate is proposed.

Notes: Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N2 fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N2 gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.

Notes: Summary The assimilation of14CO2 and [2-14C] acetate, [3-14C] pyruvate, [5-14C] α-ketoglutarate, [2,3-14C] succinate, [U-14C] glutamate and [U-14C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of14C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.

Notes: Summary By using N15-labelled calcium nitrate and ammonium sulphate, the uptake of ammonia and nitrate by young Jonathan/MM104 apple trees grown in a glasshouse in water culture was determined. A batch of 4 trees was exposed to the appropriate tracer for seven days and then sampled. This procedure was repeated at 4 weekly intervals during a 12-month period. The results show that the uptake by apple trees of either ammonia or nitrate is continuous throughout the year with a relative high peak (expressed in µg N/100 mg dry weight) in December (summer period) and low values during August-September (winter period). The total uptake in the whole tree was steady for December to June inclusive and decreased from July, with the lowest uptake during August-September period. The young trees, previously receiving nitrogen as ammonium plus nitrate (1: 7), absorbed both ions in nearly equivalent amounts, except for the winter period when the uptake of ammonia was in excess of that for nitrate. This difference in uptake of both ions probably results from a lower activity of the enzymic nitrate reducing system during the time of low temperature. Low activity of the nitrate reductase enzyme results also in a restricted uptake of nitrate by apple trees previously receiving only ammonia nitrogen. A detailed analysis of total nitrogen content showed the lowest nitrogen values (0.77%) in February, near the end of the second growth period of the first year. From then on, the total nitrogen percentage of the apple trees steadily increased to reach a peak of 1.57% N during the flush of early spring growth of the second year. Leaves, fruit, soft twig growth, blossoms and roots had the highest N levels at this time. The total nitrogen percentage of the whole tree then gradually decreased. The established distribution pattern of absorbed nitrogen from both N15-labelled compounds showed that the relatively highest incorporation of absorbed nitrogen from both sources was in newly grown tissues during early spring growth flush of the second year, indicating the importance of currently absorbed nitrogen for early spring growth. With the increase in rate of growth in the later period, the value of the nitrogen stored within the apple trees became evident.

Notes: Summary By using N15-labelled calcium nitrate and ammonium sulphate, the uptake of ammonia and nitrate by young Jonathan/MM104 apple trees grown in a glasshouse in water culture was determined. A batch of 4 trees was exposed to the appropriate tracer for seven days and then sampled. This procedure was repeated at 4 weekly intervals during a 12-month period. The results show that the uptake by apple trees of either ammonia or nitrate is continuous throughout the year with a relative high peak (expressed in µg N/100 mg dry weight) in December (summer period) and low values during August-September (winter period). The total uptake in the whole tree was steady for December to June inclusive and decreased from July, with the lowest uptake during August-September period. The young trees, previously receiving nitrogen as ammonium plus nitrate (1: 7), absorbed both ions in nearly equivalent amounts, except for the winter period when the uptake of ammonia was in excess of that for nitrate. This difference in uptake of both ions probably results from a lower activity of the enzymic nitrate reducing system during the time of low temperature. Low activity of the nitrate reductase enzyme results also in a restricted uptake of nitrate by apple trees previously receiving only ammonia nitrogen. A detailed analysis of total nitrogen content showed the lowest nitrogen values (0.77%) in February, near the end of the second growth period of the first year. From then on, the total nitrogen percentage of the apple trees steadily increased to reach a peak of 1.57% N during the flush of early spring growth of the second year. Leaves, fruit, soft twig growth, blossoms and roots had the highest N levels at this time. The total nitrogen percentage of the whole tree then gradually decreased. The established distribution pattern of absorbed nitrogen from both N15-labelled compounds showed that the relatively highest incorporation of absorbed nitrogen from both sources was in newly grown tissues during early spring growth flush of the second year, indicating the importance of currently absorbed nitrogen for early spring growth. With the increase in rate of growth in the later period, the value of the nitrogen stored within the apple trees became evident.