Dereck P. Hucklesby

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Intact preparations of plastids from pea (Pisum sativum L.) roots have been used to investigate the metabolism of glucose-6-phosphate and reduction of inorganic nitrite within these organelles. The ability of hexose-phosphates to support nitrite reduction was dependent on the integrity of the preparation and was barely measurable in broken organelles. In intact plastids, nitrite was reduced most effectively in the presence of glucose-6-phosphate (Glc6P), fructose-6-phosphate and ribose-5-phosphate and to a lesser extent glucose-1-phosphate. The Km (Glc6P) of plastid-located Glc6P dehydrogenase (EC 1.1.1.49) and Glc6P-dependent nitrite reduction were virtually identical (0.68 and 0.66 mM respectively) and a similar relationship was observed between fructose-6-phosphate, hexose-phosphate isomerase (EC 5.3.1.9) and nitrite reduction. The pattern of release of CO2 from different carbon atoms of Glc6P supplied to root plastids, indicates the operation of both glycolysis and the oxidative pentose-phosphate pathway with some recycling in the latter. During nitrite reduction the evolution of CO2 from carbon atom 1 of Glc6P was stimulated but not from carbon atoms 2, 3, 4, or 6. The importance of these results with regard to the regulation of the pathways of carbohydrate oxidation and nitrogen assimilation within root plastids is discussed.

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor).

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.

Physical and kinetic properties of photosynthetic phosphoenolpyruvate carboxylase in developing apple fruit

Abstract Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Coxs Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H 14 CO 3 − incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent K m (PEP) and V max values compared more favourably with those obtained from a C 3 -species (spinach) than from a C 4 -species (maize). l -Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C 4 photosynthetic enzyme.

Stability of the nitrosyl-sirohaem complex of plant nitrite reductase, investigated by EPR spectroscopy.

Nitrite reductase (nitrite:ferredoxin oxidoreductase EC 1.7.7.1) is an enzyme that contains one sirohaem group [l-3] and one iron-sulphur cluster [2,7] per molecule. The substrate nitrite and other nitrogenous intermediates of the reaction are presumed to bind to the sirohaem. Electron paramagnetic resonance (EPR) spectroscopy showed that in the isolated enzyme, the sirohaem was in the high-spin state. On treatment with dithionite and nitrite, a new signal appeared at g = 2.007, 2.058 [2]. This was presumed to arise from an intermediate in the enzyme reaction as it was observed in samples frozen under turnover conditions [4]. The signal was assigned to an Fe(II)-NO complex of sirohaem which would represent the first stage in the reduction of nitrite to ammonia. In the earlier observations, 14N hyperfine structure which would have corroborated this interpretation was not observed. Certain nitrosyl complexes of haem proteins are known not to give resolved 14N hyperfine structure in their powder spectra unless modified in some way. For example, in haemoglobin nitrosyl the splitting is not observed unless the protein structure is perturbed by sodium dodecyl sulphate [5]. Closer examination of the EPR spectrum of nitrosyl-sirohaem nitrite reductase has revealed hyperfine structure. We present here details of the spectra derived from 14NO; and “NO;. The different hyperfine patterns in the spectra of these two complexes made it possible to investigate the possibility of exchange of bound NO. It was concluded that the complex is very stable and exchange was not detectable unless the enzyme underwent turnover.

Cyanide formation from glyoxylate and hydroxylamine catalysed by extracts of higher-plant leaves

Extracts of spinach, maize and barley contain an enzyme which catalyses the formation of hydrogen cyanide from glyoxylate and hydroxylamine. The enzyme is dependent upon ADP and a divalent cation such as manganese. Glyoxylicacid oxime is a poor substrate for the enzyme. Carbon dioxide is another product of the reaction and is probably produced in 1:1 stoichiometry with hydrogen cyanide. The possible relationship of this enzyme to the regulation of nitrate reduction is discussed.

Distribution and Physiological Significance of Photosynthetic Phosphoenolpyruvate Carboxylase in Developing Apple Fruit

Summary Phosphoenolpyruvate carboxylase was extracted from hypodermal and vascular tissue and seeds during the development of apple fruit cvs. Golden Delicious and Coxs Orange Pippin. For all three tissues the enzyme activity per whole fruit increased throughout the growing season and disproportionated in favour of seeds in September and October. Enzyme activity per fruit increased to assimilate from 0.01 mg C0 2 h −1 in June to 10 mg C0 2 h - −1 in October when the C0 2 reassimilation potential of the fruit rose to exceed its C0 2 loss by several fold.

Metabolism of hydroxylamine by spinach leaf discs and its relationship to nitrate reduction

Nitrate reduction in vivo by spinach leaf discs was shown to be inhibited by hydroxylamine when this was included in the nitrate reductase assay solutions or introduced to the tissue during a preincubation period. The sensitivity of nitrate reduction to hydroxylamine was not sufficient to suggest a natural process, considering the small endogenous concentrations of hydroxylamine in the leaves. Inhibition of nitrate reduction in vivo could be approximately related to rates of in vitro inhibition of nitrate reductase by this compound. There was no need to suppose conversion of hydroxylamine to cyanide to inhibit nitrate reduction. Some of the in vivo and in vitro characteristics of hydroxylamine inhibition of nitrate reductase are described. Hydroxylamine was metabolised by discs at rates comparable to nitrate reduction. Rates of metabolism of hydroxylamine, and its accumulation in the tissues from an external solution were both enhanced by light but little affected by anaerobiosis.

Utilization of bicarbonate by apple fruit Phosphoenolpyruvate carboxylase

Abstract Phosphoenolpyruvate carboxylase (PEPC), was partially purified from apple fruit cv Golden Delicious. Kinetic values for PEP and HCO − 3 suggest a capacity for efficient carbon dioxide refixation. PEPC activity was maximal between 5–10 mM carbonate (HCO − 3 ) and inhibition was observed above 10 mM HCO − 3 . In conditions of PEP-saturation, HCO − 3 inhibition of apple fruit PEPC activity appeared non-competitive with respect to PEP and was partially reversible.