The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMOm), using the non-ionic detergent dodecyl-β-d-maltoside, to yield solubilized enzyme (pMMOs). Further purification gave rise to an active complex (pMMOc) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24kDa subunits) and the pMMOR (63 and 8kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with 63Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe3+ ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.

Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes

Phosphatidylethanol is formed by ‘transphosphatidylation’ of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca2+-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca2+-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidylalcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca2+-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca2+ concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca2+-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

Properties of chicken erythrocyte histone deacetylase associated with the nuclear matrix

Histone H2B is deacetylated more rapidly than H3 and H4 in chicken immature erythrocytes. Histone deacetylase from chicken immature erythrocytes was partially purified, and the histone specificities of the multiple histone deacetylase forms were determined. Ion-exchange (Q-Sepharose) and gel-exclusion (Superdex 200) chromatography of extracts from erythrocyte nuclei showed two forms (HD1 and HD2) of histone deacetylase. HD1, with a molecular mass of about 55 kDa, preferred free H3–H4 relative to H2A–H2B, while HD2, with a molecular mass of approx. 220 kDa, had a slight preference for H3–H4. HD1 and HD2 differed in pH- and ionic-strength-dependence. HD2 dissociated into HD1 when treated with 1.6 M NaCl or when applied to a Q-Sepharose column. The enzymic properties of nuclear-matrix-bound histone deacetylase showed a striking difference from that of HD1 and HD2, particularly in its strong preference for H2A–H2B. Treatment of the nuclear matrix with 1.6 M NaCl and 1% 2-mercaptoethanol solubilized histone deacetylase, which chromatographed as 400 and 220 kDa forms on a Superdex 200 column. The solubilized enzyme retained its histone preference for H2A-H2B. Chromatography of the nuclear-matrix-derived enzyme on Q-Sepharose yielded one peak of enzyme activity with chromatographic properties and histone specificities similar to those of HD1. These results provide support for the active form of the enzyme in situ being a high-molecular-mass complex associated with proteins that are components of the nuclear matrix. Substrate preference of the enzyme is governed by the proteins associated with the histone deacetylase.

The anticalmodulin effect of aflatoxin B1 on purified erythrocyte Ca2+-AtPase

The genotoxic carcinogen aflatoxin B1 (AFB1) inhibited the calmodulin-stimulated membrane-bound (Ca2+Mg2+)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB1 caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca2+-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca2+-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB1 did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB1 inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB1 inhibits the activated Ca2+-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca2+-calmodulin or acidic phospholipids. The implication of these observations is that Ca2+-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB1 because of its anticalmodulin effect.

131 PEROXIDATIVE CHALLENGE OF PLASMA MEMBRANE VESICLES FROM BELL PEPPER FRUIT LEADS TO DECREASED H+ATPase ACTIVITY

-Lipid peroxidation has been proposed as an important factor in chilling injury of susceptible fruits and vegetables. The effect of in vitro peroxidative challenge on H+ATPase activity in intact plasma membrane vesicles and solubilized enzyme was determined by incubation with (1) deionized water (control), (2) Fe3+-ascorbate, and (3) lipoxygenase (LOX) + phospholipase A2(PLA2) for 0, 30, and 60 min. Enzyme activity increased throughout the incubation period with no accumulation of thiobarbituric acid-reactive substances (TBA-RS) in the control, but vesicles challenged by the peroxidative systems showed significant increases in TBA-RS and decreases in membrane-bound H+ATPase activity. Greater losses in H+ATPase activity were observed in solubilized enzyme than in intact vesicles. The results indicate that loss of H+ATPase activity due to chemical modification of the protein rather than changes in membrane fluidity and suggest that modification is away from the active site.