The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

The mechanism of deuterium oxide-induced protein degradation in Lemna minor

Transfer of Lemna minor fronds to culture medium containing 50% (v/v) deuterium oxide induces a large increase in the rate of protein breakdown, which is not due to an increase in the activity of acidic or neutral proteolytic enzymes or peptidases. Biochemical and ultrastructural evidence indicates that deuterium oxide affects the properties of certain membranes, particularly the tonoplast, and allows vacuolar proteolytic enzymes to pass into the cytoplasm and cause the increased protein breakdown.Abbreviations BAPA benzylarginine-p-nitroanilide - LPA leucine-p-nitroanilide - TCA trichloroacetic acid     

Composés glucidiques et azotés et résistance à la sécheresse chez Ailanthus altissima

In Ailanthus altissima, carbohydrate and protein reserves are mainly localized in the tap-root. Under drought stress they are very quickly hydrolysed. Despite the water deficiency, an increase in starch synthesis is observed in the leaves and stem during the first stages of stress. Stimulation of the cambial activity of the stems and an increase in protein content have also been observed. Chemical analyses have shown a high proline content which increases markedly following drought stress, especially in the tap-root.     

Passive cable properties of hippocampal CA3 pyramidal neurons

The passive electrical cable properties of CA3 pyramidal neurons from guinea pig hippocampal slices were investigated by applying current steps and recording the voltage transients from 25 CA3 neurons, using a single intracellular microelectrode and a 3-kHz time-share system. Two independent methods were used for estimating the equivalent electrotonic length of the dendrites, L, and the dendritic to somatic conductance ratio, . The first method is similar to that used by Gorman and Mirolli (1972) and gave an average L of 0.96; the average was 2.44. The second method is derived here for the first time and assumes a finite-length cable with lumped soma. It is an exact solution for L and , using the slopes and intercepts of the first two peeled exponentials. The average L was 0.94; the average was 1.51. The results, using both methods, are in close agreement. The average membrane time constant for all 25 CA3 neurons was 23.6 ms, suggesting a large (23,600 cm²) average membrane resistivity. It is concluded that CA3 neurons are electronically short.This work was supported by Grants NS 11535 and NS 15772 from the National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, U.S. Public Health Service.     

Purification and properties of O-acetyl-L-serine sulphydrylase from wheat leaves

O-Acetyl-L-serine sulphydrylase (OASS), the enzyme which produces L-cysteine from O-acetyl-L-serine (OAS) and sulphide, is     

Starch breakdown in the spadix of Arum maculatum

The aim of this work was to discover the pathway of starch breakdown during thermogenesis in the club of the spadix of Arum maculatum. The conventional α-amylase of higher plants could not be demonstrated in extracts of clubs although such extracts did exhibit considerable hydrolytic activity towards starch. This activity had an action pattern characteristic of an endo-amylase, was destroyed by heating to 70°, and was not inhibited by either 7 mM ethylenediaminetetra-acetic acid or 100 mM N-ethyl maleimide. Measurements of this hydrolytic activity, and of the maximum catalytic activities of starch phosphorylase, phosphoglucomutase and hexokinase, were made at different stages of club development. These measurements were compared with estimates of the rate of starch breakdown at thermogenesis. This comparison indicates that phosphorolytic cleavage does not play a large role in such starch breakdown, and that this process is mediated, mainly, by the hydrolytic activity, described above, and by hexokinase.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

An aminopeptidase from Agave americana thermodynamic studies

Purified Agave aminopeptidase was characterized with respect to the thermodynamic properties of the reaction catalysed by the enzyme. Kinetic studies were conducted at different temperatures ranging from 7.8 to 45°. The energy of activation, E_a, as well as the constants ΔH, ΔF and ΔS were calculated for both the formation of the enzyme-substrate complex, and the dissociation of the enzyme-product complex. Kinetic studies in buffers with varying dielectric constants enabled the determination of the electrostatic as well as the non-electrostatic components of ΔS. These results fit well into the overall kinetic picture of this enzyme-catalysed reaction as reported previously.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Purification and properties of mannitol dehydrogenase from Agaricus bisporus sporocarps

Mannitol dehydrogenase (mannitol: NADP⁺ 2-oxidoreductase: EC 1.1.1.138) was isolated from Agaricus bisporus by fractionation with protamine sulphate and (NH₄)₂SO₄, followed by chromatography on DEAE-Sephadex, then by affinity and gel chromatography. The products of enzyme reaction were identified by GLC and TLC. K_m, optimum pH, MW and pI of the enzyme as well as the influence of temperature, ions and inhibitors on enzymic activity were determined. In the sugar reducing reaction, the enzyme was specific for fructose but, in the reverse direction, some structurally related polyols could substitute for mannitol. The enzyme was very sensitive to alterations in the NADP⁺/NADPH ratio. The results are discussed in relation to the possible role of mannitol dehydrogenase in fungal metabolism.