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The ras family of oncogenes, as well as the transplasma membrane electron transport system, have been implicated in cellular growth control. Here we present evidence that the Ha-ras oncoprotein increases the activity of the transplasma membrane electron transport system in the mouse embryonic fibroblast cell line C3H10T1/2. Both cytochrome c and ferricyanide are reduced at a faster rate by C3H10T1/2 cells which are expressing the Ha-ras oncogene. In addition, the Ha-ras transformed cells extrude protons faster in response to stimulation by external oxidants than their normal counterparts. These results suggest that transformation events initiated by the expression of the Ha-ras oncoprotein, p21, in C3H10T1/2 cells have a controlling effect on the transplasma membrane electron transport system.

Long-term treatment with ethidium bromide of HL-60 cells induced a mitochondria-deficient ρ° cell line, where mitochondrial DNA can not be identified by PCR and cytochromecoxidase activity was 80% decreased. These cells showed a progressive increase of ascorbate stabilization which was 52% higher in the established ρ°HL-60 cells. Both CoQ10and NADH-ascorbate free radical reductase of the plasma membrane were increased in ρ° HL-60 cells compared to parental cells, while NADH-cytochromecreductase was unchanged. CoQ10is a component of the ascorbate stabilization activity in the plasma membrane that would provide both a mechanism to deplete the excess of NADH produced in ρ° HL-60 cells and for resistance to oxidative stress.

Albright hereditary osteodystrophy (AHO) is characterized by multiple somatic defects secondary to mutations in the GNAS1 gene. AHO patients with mutations on maternally inherited alleles are resistant to multiple hormones (e.g., PTH, TSH), a variant termed pseudohypoparathyroidism (PHP) type 1a, due to presumed tissue-specific paternal imprinting of the a chain of Gs as demonstrated in murine renal proximal tubule and fat cells. Studies in human tissues thus far revealed imprinting only in pituitary. Because mild hypothyroidism due to TSH resistance occurs in most PHP type 1a patients, we investigated whether Gas is imprinted in thyroid. Examination of eight normal thyroids demonstrated significantly greater expression from the maternal GNAS1 allele, with paternal Gas transcripts accounting for only 25.9–40.4%. Expression of NESP55, XLas, and 1A was uniallelic. We conclude that Gas is incompletely imprinted in the thyroid, which provides an explanation for mild TSH resistance in PHP type 1a. 2002 Elsevier Science (USA). All rights reserved.

Long-term treatment with ethidium bromide of HL-60 cells induced a mitochondria-deficient rho degree cell line, where mitochondrial DNA can not be identified by PCR and cytochrome c oxidase activity was 80% decreased. These cells showed a progressive increase of ascorbate stabilization which was 52% higher in the established rho degree HL-60 cells. Both CoQ10 and NADH-ascorbate free radical reductase of the plasma membrane were increased in rho(0)HL-60 cells compared to parental cells, while NADH-cytochrome c reductase was unchanged. CoQ10 is a component of the ascorbate stabilization activity in the plasma membrane that would provide both a mechanism to deplete the excess of NADH produced in rho(0)HL-60 cells and for resistance to oxidative stress.

A 34 kDa coenzyme Q reductase has been solubilized and purified from pig liver plasma membranes. The solubilized enzyme reduced coenzyme Q0 with NADH. Ubiquinones with longer isoprenoid side chain such as Q2 and Q10 were also reduced when the quinones and the enzyme were reconstituted into phospholipid liposomes. N-terminal sequencing of an internal peptide showed identity to bovine NADH-cytochrome b5 reductase. Biochemical characterization of the purified enzyme indicated that the coenzyme Q reductase corresponds to an unusual form of NADH-cytochrome b5 reductase.

Treatment of CCI 39 cells with the impermeable iron II chelator bathophenanthroline disulfonate (BPS) inhibits both DNA synthesis and transplasma membrane electron transport. The inhibition persists when the BPS is removed, and the extract from 106 cells contains up to 1.28 nmoles iron II chelated to BPS. The BPS iron II chelate itself is not inhibitory. Both DNA synthesis and electron transport are restored by addition of μM iron II or iron III compounds to extracted cells. Other impermeable chelators for iron II give similar inhibition, whereas the iron III-specific Tiron or copper-specific bathocuproine sulfonate do not inhibit. The inhibition differs from the permeable iron III chelator inhibition of ribonucleotide reductase, because inhibition of DNA synthesis by the permeable chelators is reversed when chelator is removed. The response to growth factors also differs, with no impermeable chelator inhibition on 10% fetal calf serum contrasting to inhibition by permeable chelators. DNA synthesis with both activation of tyrosine kinase with EGF plus insulin or by thrombin or ceruloplasmin led to protein kinase C activation as inhibited by the impermeable chelators. It is proposed that an iron available on the cell surface is required for DNA synthesis and plasma membrane electron transport.

Coenzyme Q added to culture media stimulates the growth of HeLa and Balb/3T3 cells in serum free conditions. The stimulation by coenzyme Q is additive to the stimulation by ferricyanide, an impermeable electron acceptor for the transplasma membrane electron transport. αTocopherylquinone can also stimulate cell growth, but vitamin K1 is inactive or inhibitory. The response to coenzyme Q and ferricyanide is enhanced with insulin. A contribution to plasma membrane NADH oxidation or modification of the membrane quinone redox balance can be a basis for the growth stimulation.

NADH oxidation by pig liver plasma membranes is stimulated by ceruloplasmin (CUP) reaching a maximal value at 50 U/ml of CUP. NADH oxidation activated by CUP is proportional to the amount of protein. Concanavalin A (Con A) which recognizes the glucidic residues of the CUP required for binding to the receptor inhibits the NADH oxidation in a dose-responsive manner. Both adriamycin and bathophenantroline disulfonate (BPS), previously reported as transplasma membrane electron transport inhibitors, also inhibit the CUP-stimulated NADH oxidation of pig liver plasma membranes. Our results show a clear interaction between CUP and the NADH oxidase of plasma membrane, which supports an oxidative role for CUP in its growth effect.

The incorporation of tritiated thymidine into CCL-39 cells grown in the absence of fetal calf serum or other growth factors is greatly increased by low concentrations of ceruloplasmin. The stimulation is greater than observed with serum or thrombin. Addition of serum decreases the thymidine incorporation with ceruloplasmin to the level with serum alone. As with serum, the response to ceruloplasmin is high at both 20% and 1% oxygen, which is consistent with the action of ceruloplasmin as an oxidant with a high affinity for oxygen. Since transplasma membrane electron transport increases cell growth and thymidine incorporation, ceruloplasmin may act as a terminal oxidase for ferrous iron or ascorbate to stimulate transplasma membrane electron transport. The four electron transfer from ceruloplasmin to oxygen to form water will prevent peroxide formation at the cell surface. Alternatively, superoxide formation inside the cell or membrane could employ the superoxide dismutase function of ceruloplasmin to produce peroxide. Either mechanism would be consistent with the previously described stimulation of growth by external oxidants.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.

Interferon inhibits uptake of the radiolabeled queuine analog, rQT3, into cultured human fibroblasts. Simultaneous exposure to 10 nM phorbol-12, 13-didecanoate (PDD) potentiates interferon-induced inhibition of rQT3 into cultured fibroblasts. All three major classes of human interferon tested affected uptake similarly, with fibroblasts derived beta-interferon being more effective in dose response than gamma or alpha interferons. This suggests that endogenous production of interferon by cultured cells, such as that observed during a low grade viral infection, inhibits queuine uptake and may subsequently lead to a decreased level of queuine modified transfer RNA. Queuine-hypomodified transfer RNA has been implicated in growth control, differentiation and neoplastic transformation.

Protein kinase C modulates the activity of a highly specific uptake mechanism for queuine in cultured human fibroblasts. Activators of protein kinase C induce an increased uptake rate for the radiolabeled analog of queuine, rQT3. The protein kinase C inhibitors, H-7, staurosporine and sphingosine all induced a dramatic decrease in the uptake rate of rQT3. This suggests that protein kinase C is tied to efficient cellular uptake of queuine. Uptake is prerequisite to the modification of transfer RNA with queuine. Perturbation of queuine-modified transfer RNA levels has been associated with neoplastic transformation, differentiation and growth control.

2,6-dibromothymoquinone (DBMIB) and other coenzyme Q analogs partially inhibit electron transport and the membrane-bound Mg++ stimulated ATPase of E. coli membranes. The inhibitions by DBMIB are fully reversed by coenzyme Q6, and other analogs show partial reversal by coenzyme Q6. Electron transport reactions inhibited are NADH and lactate oxidase, NADH menadione reductase, lactate phenazinemethosulfate reductase and duroquinol oxidase. The concentrations of DBMIB required are similar for electron transport and ATPase inhibition and inhibitions are all increased by uncouplers. Electron transport and ATPase are not inhibited in a DBMIB insensitive mutant. Soluble ATPase extracted from the membranes does not show DBMIB inhibition under either high or low Mg++ conditions. Lipophilic chelators show additional inhibition over DBMIB. It appears that coenzyme Q functions at three sites in E. coli electron transport where ATPase activity is controlled. Coenzyme Q deficient mutants also show decreased electron transport and ATPase activity which is restored by coenzyme Q.

Electron transport across the plasma membrane is found in all cells which have been tested. This activity has been implicated in control of cellular growth, transport and hormone response. In virus transformed cells and tumor cells we find the activity is decreased and becomes sensitive to the antitumor drug adriamycin. Inhibition of transmembrane redox by adriamycin parallels cytoxicity to transformed cells.

It was found that platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) inhibits electron transport greater than 90% in Photosystem II of spinach chloroplasts in concentrations from 2.8 to 3.5 micrograms/ml. The inhibition of the main pathway of electron transport through Photosystem II appeared to be specific for the platelet activating factor. Phorbol myristate acetate, 1,2-dipalmitin or fatty acid esters gave 17-32% inhibition in higher concentrations. The inhibition site for platelet activating factor was localized close to the reaction center of Photosystem II, based on the inhibition of the donor reaction, diphenyl carbazide----indophenol, in Tris-treated chloroplasts. Other Photosystem II reactions, H2O----silicomolybdic acid, H2O----2,5-dimethylbenzoquinone, or H2O----indophenol, were also inhibited by platelet activating factor. The present data point out the unique inhibition of Photosystem II electron transport by the platelet activating factor, but do not support the operation of a phosphoinositide cycle in chloroplasts.

The effect of various chelators (orthophenanthroline, bathophen-anthroline, bathophenanthroline sulfonate and bathocuproine) on electron transport of spinach chloroplasts has been studied by means of various photosystem I and II reactions. It was found that photosystem II has at least 3 chelator-sensitive sites, photosystem I from 3–4. An uncoupler-affected site was found in each photosystem. In addition, photosystem I had a stimulator site and a soak site. The soak site was sensitive to chelators only after a period of incubation with the chelator.

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.

The oxygen evolution associated with electron transport in spinach chloroplasts can be stimulated by low concentrations (

Summary An abbreviated Photosystem II electron transport sequence from water to silicomolybdate plus ferricyanide functions in the presence of DCMU. This electron transfer was not coupled to photophosphorylation, and therefore places phosphorylation associated with Photosystem II (Site II) after the site of DCMU inhibition. Silicomolybdate per se was not inhibitory to phosphorylation in either the cyclic or noncyclic (water to methylviologen) electron transport mode.

Summary Both the positive vanadyl and negative vanadite ions are oxidized by mitochondria or purified cytochrome c oxidase. The oxidation does not require cytochrome c and is not inhibited by the cytochrome c antagonist polylysine. Oxidation rates for vanadyl compare well with rates for cytochrome c oxidation. Since the oxidation is not inhibited by azide, cyanide and carbon monoxide it does not appear to be catalyzed by the cytochrome a3 pathway. Inhibition by diphenylthiocarbazone and salicylaldoxime indicate that one of the copper sites in cytochrome oxidase can be the site of oxidation.

Summary At low Mg ++ concentration the soluble Mg ++ /Ca ++ activated ATPase extracted from E. coli membranes is inhibited by several chelators including bathophenanthroline sulfonate, diphenylthiocarbazone and zincon. Incubation of the enzyme with zincon shows a gradual extraction of zinc. Activity is lost in proportion to zinc removal and is specifically restored by readdition of zinc to the extracted enzyme. The soluble enzyme in presence of high concentration of Mg ++ is not inhibited by chelators. The presence of high Mg ++ concentration also prevents reconstitution of the zinc depleted enzyme activity with zinc. In the membrane bound form the activity is not inhibited by zincon or other water soluble chelators regradless of Mg ++ concentration whereas the activity is inhibited by various lipophilic chelators such as thenoyltrifluoroacetone and tert-octyl catechol. These latter chelators do not inhibit the soluble ATPase. We conclude that the ATPase contains zinc which is protected from chelators by high Mg ++ concentration or by binding to the membrane and that there is another lipophilic chelator site in the membrane which also controls activity of the membrane-bound ATPase.

Summary The use of water soluble bathophenanthroline sulfonate as an inhibitor of electron transport in mitochondria and electron transport particles exhibits effects that may be related to the location of the multiple non-heme iron and copper proteins in the membrane. In contrast to lipophilic bathophenanthroline, hydrophilic bathophenanthroline sulfonate gives inhibition dependent upon membrane orientation. While NADH oxidation is sensitive in both intact mitochondria (C-side exposed) and electron transport particles (M-side exposed), succinate oxidation is inhibited only in mitochondria. Duroquinol oxidase is stimulated in mitochondria but unaffected in electron transport particles. Inhibitions of NADH and succinate by electron acceptor such as juglone, ferricyanide, duroquinone, and cytochrome c in the presence of appropriate inhibitors indicate at least one chelator-sensitive site on each side of the membrane for the NADH dehydrogenase complex and at least one site on the C-side in the succinate dehydrogenase complex. In contrast, lipophilic bathophenanthroline shows substantial inhibition at these and additional sites regardless of membrane orientation. A transmembranous orientation of dehydrogenase components is proposed having both surface exposed and buried chelator sensitive sites.

Selective inhibition of ferricyanide reduction in photosystem II by lipophilic thiols indicates a unique pathway of electron transport, which is not involved in reduction of class III acceptors or transfer of electrons to photosystem I. Both aromatic and aliphatic thiols induce the inhibition, but thiol binding reagents such as p-hydroxymercuribenzoate or N-ethylmaleimide do not inhibit. The inhibition can be observed using either dibromothymoquinone or bathophenanthroline to direct electrons away from photosystem I. No pretreatment of chloroplasts with thiols in the light was necessary to inhibit ferricyanide reduction by photosystem II or the O2 evolution associated with ferricyanide reduction.

The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.

The effects of the inhibitors antimycin, 2-heptyl-4-hydroxyquinoline N-oxide (NOQNO) and 2-nonyl 4-hydroxyquinoline N-oxide (NQNO) on the cytochrome b-c 1 region spectrum of submitochondrial particles are compared with those of a new inhibitor 2-ω-cyclohexyl pentyl 3-hydroxy 1,4-naphthoquinone. Dithiothreitol without electron mediators can cause selective reduction of part of the cytochromes b. Evidence for at least four cytochromes b is presented.

A number of carbonyl compounds including bicarbonate, ethylene carbonate, dimethylcarbonate, propylene carbonate, bis-pentamethylene urea, and glycidol, and several chelators were tested for their effect on photosynthetic reactions in isolated spinach chloroplasts. It was found that carbonyl compounds inhibited the DCMU-insensitive silicomolybdate reduction by photosystem II but stimulated the O 2 evolution associated with ferricyanide reduction in presence of DBMIB and the H 2 O→methylviologen reaction. Many chelators behaved in the same manner except 1,10-phenanthroline which shows the opposite effect. The carbonyl compounds did not uncouple because they stimulated the proton gradients associated with noncyclic photophosphorylation, whereas some chelators, such as bathocuproine or bathophenanthroline inhibited the proton gradients 100%. Electron transport in presence of ADP and inorganic phosphate showed a stimulation of rates beyond that obtained in presence of an uncoupler. The data are discussed in terms of inhibition of cyclic electron flow around PS II which leads to increased electron transport rates toward PS I.

Highly purified mouse liver plasma membranes have been used to define the properties of an NADH dehydrogenase activity associated with plasma membrane. The NADH indophenol reductase activity is two-fold stimulated at 5 × 10−8 M glucagon and the stimulation is inhibited by atebrin. Corresponding activity in endoplasmic reticulum is not stimulated by glucagon. The NADH indophenol reductase is 90% inhibited by insulin at 7 × 10−11M and shows a return to the original activity at higher insulin concentrations. NADH dehydrogenase activity in endoplasmic reticulum is inhibited up to 50% by insulin at a similar concentration. Triiodothyronine at 10−7M also inhibits the plasma membrane dehydrogenase whereas thyroxine has little effect. The response of this dehydrogenase to hormones suggests a role in regulation of cellular function.

Previous attempts by Wainio and Aronoff (1955) and Yonetani (1959) to solubilize cytochrome oxidase with non-ionic surfactants were reported as unsuccessful. Their failure to achieve positive results can be attributed to the remarkably critical structure of the surfactant required to effect the solubilization and the critical conditions under which the process must be carried out. In this communication we describe the procedure by which Triton X-100 (9–10 oxyethylene groups) in the presence of .2M neutral potassium phosphate effects the complete solubilization of the cytochrome oxidase protein from the membraneous oxidase preparation described in the accompanying communication.

The positioning of fatty acids within cardiolipin from beef heart mitochondria was determined and compared to phosphatidyl choline and phosphatidyl ethanolamine; from the same source. Unsaturated fatty acids were preferentially esterified in the β-position and saturated fatty acids were contained preferentially in the α-position of the choline and ethanolamine phosphatides. In contrast, fatty adds were nearly randomly distributed between the (α- and β-positions of cardiolipin. On acetolysis, cardiolipin yielded diglyceride acetates which were separated into three distinct species, trienes, tetraenes and pentaenees, by argentation thin-layer chromatography. Tetraenes accounted for more than 75% of the total diglyceride acetates and contained 94% linoleic acid, suggesting the existence of a molecular species of cardiolipin containing solely linoleic acid.

Quinones can be used as electron acceptors and donors in a new approach to the study and resolution of the respiratory chain-linked NADH dehydrogenase system of beef heart mitochondria. Juglone (5-hydroxynapthoquinone), ubiquinone, duroquinone (tetramethylbenzoquinone), and ferricyanide are found to react at four separate sites in the complex. The sites can be defined by chemical and enzymatic inhibition or by fractionation of the system. The reduced form of one quinone, duroquinol, has the unique property of being an electron donor for the energy-linked reduction of NAD+.

The presence of an amytal-sensitive NADH-menadione reductase in beef heart mitochondria has been previously reported (Cunningham, et al., 1965; Cunningham, et al., 1965b; Crane, et al., 1965). The enzyme described was found in the aqueous supernatant after extraction of beef heart mitochondria with diethyl ether. The enzyme has been further studied and purified and has been found to undergo changes during purification procedures, as shown by its response to different inhibitors. We present data which describe some of the characteristics of the enzyme and which suggest the enzyme is related to the electron transport chain activity in mitochondria.

Cytochrome a,a3 has been detected in highly purified nuclear membrane preparations. Nuclear membranes resemble microsomal membranes in cytochrome b5 content but differ by the absence of the microsomal cytochrome P-450. Cytochrome a,a3 content in nuclear membranes is 30% that of mitochondria, and cytochrome b5 content is 33% of control microsomes.

A small amount of cardiolipin (diphosphatidyl glycerol) is tightly bound to cytochrome oxidase. The stoichiometry approaches one molecule of cardiolipin per molecule of cytochrome a. Solvent extraction and phospholipase treatments which remove or destroy other phospholipids and the bulk cardiolipin do not effect this tightly bound cardiolipin. Potential oxidase activity and membrane formation potential are lost when the tightly bound cardiolipin is removed. This cardiolipin is very similar in fatty acid composition to the total mitochondrial cardiolipin.

Heme a protein separated from a purified cytochrome oxidase preparation shows an unexpectedly low molecular weight of 26,500 by gel electrophoresis. The tightly bound phospholipid, copper and iron are associated with this low molecular weight protein. This protein also retains the capacity to form membrane sheets when associated with phospholipid. The other protein separated from the oxidase has a higher molecular weight 55,000, no phospholipid, copper or iron and does not form membrane layers when phospholipid is added. It can be tentatively identified as core protein IV. Some loss of oxidase activity occurs when the two proteins are separated which can be partially restored by recombination of the fractions.

Summary Actinomycin D and puromycin inhibit the NADH dehydrogenase activity of mouse liver and pig erythrocyte plasma membrane. Actinomycin is effective at 10−6 M and inhibits the transmembrane ferricyanide reduction by whole erythrocytes. The NADH dehydrogenase of endoplasmic reticulum is not inhibited. Puromycin inhibits the plasma membrane dehydrogenase but does not inhibit ferricyanide reduction by whole erythrocytes. Effects of these drugs on metabolism after short term exposure may be mediated through the plasma membrane dehydrogenase.

Summary The lipophilic iron chelators, bathophenanthroline and TTFA inhibit the membrane-bound light-activated and sulfhydryl-pendent Mg ++ -ATPase of spinach chloroplasts, but not the trypsin-activated Ca ++ -ATPase of the solubilized enzyme. Phosphorylation was also inhibited by both chelators. Under these conditions BP acts as an electron transport inhibitor, not an energy transfer inhibitor, i.e. the inhibition was not reversed by uncouplers. TTFA on the other hand showed the classical energy transfer inhibition pattern, the inhibition being reversed by uncouplers. The chelators also make the membranes more leaky toward H + ions. This alone does not seem to account for the inhibition of energy transduction, i.e. the chelators are not simply uncouplers. The data are consistent with the concept that a metal ion may be intimately involved in energy transduction.

Oligomycin sensitive, membrane bound ATPase of beef heart mitochondria is strongly inhibited by the lipophilic chelator bathophenanthroline. The inhibition is reversed by uncouplers such as carbonyl-cyanide-3-chlorophenyl hydrazone but not by ionophores such as gramacidin. Oligomycin-insensitive soluble ATPase is not inhibited by bathophenanthroline. Since the inhibition effects parallel bathophenanthroline inhibition of electron transport associated with coupling sites and uncoupler reversal is similar we propose metalloproteins function at the juncture of the electron transport and energy coupling systems.

Impermeable ferricyanide stimulates the growth of HeLa cells in absence of fetal bovine serum or other growth factors. A series of impermeable oxidants with redox potentials down to −125 mV stimulate equivalent growth. All of these oxidants are reduced by the transplasma membrane electron transport system. Oxidants with redox potentials below −175 mV are not reduced by the transmembrane electron transport and do not stimulate growth. Insulin which stimulates growth in absence of serum also stimulates transmembrane ferricyanide reduction. Ferricyanide increases growth in presence of insulin. Antitumor drugs, which inhibit HeLa cell growth, inhibit the transplasma membrane redox system. Transplasma membrane electron transport is accompanied by proton release from HeLa cells.

Plasma membrane isolated by two-phase partition from rat liver showed rates of ascorbate free radical reduction by NADH of 4–5 nmoles of oxidized NADH/min/mg protein. This activity was inhibited 80% by ConA and up to 97% by WGA and LFA lectins. NADH-ascorbate free radical reductase was also inhibited in rat liver plasma membranes preincubated with neuraminidase or trypsin, but no additional inhibition was observed in the presence of LFA after enzyme digestion. It appears that the integrity of glucan moieities of the cell surface glycoconjugates are necessary for the optimal function of this activity that could be considered as part of the transplasma membrane electron transport system.

Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.

Inhibition of coupled electron transport in mitochondrial cristae by hydrophobic chelators such as bathophenanthroline is partially prevented by uncoupling agents such as carbonylcyanide-m-chlorophenyl hydrazone. Reversal of inhibition is observed in all three sections of the electron transport chain where energy coupling occurs. Sites inhibited by hydrophilic bathophenanthroline sulfonate or orthophenanthroline do not show uncoupler relief of inhibition. We conclude that a buried non-heme iron or copper protein is closely associated with each energy coupling site.

NADH semidehydroascorbate oxidoreductase activity is present in clathrin coated vesicles isolated from rat liver. The activity of the enzyme on the coated vesicles and Golgi apparatus but not that of endoplasmic reticulum is stimulated by calmodulin and is inhibited by three different drugs which are known inhibitors of calmodulin function including trifluoperizine, pimozide and R24571. Extraction of clathrin from the vesicles causes a decrease in activity which can be partially restored when the extracted clathrin is added back. Added calmodulin also restores much of the activity which is lost when the clathrin is removed and the specific activity of added pure calmodulin is similar to that of the crude clathrin on a protein basis. There is a decrease in enzyme activity if coated vesicles or Golgi apparatus are treated with a calcium antagonist (8-[N,N-diethylamino]-octyl-3,4,5-trimethoxybenzoate) (TMB-8). However, the enzyme activity can be recovered to that of the untreated control if calcium (6.0 mM) is added. An additive stimulatory effect on enzyme activity is also observed when both calcium (1.0 mM) and calmodulin (40 micrograms/ml) are present in the vesicles simultaneously. The results show that the NADH-semidehydroascorbate oxidoreductase of coated vesicles and Golgi apparatus have regulatory properties different from those of the microsomal electron transport system. Calmodulin-calcium control mediated through the semidehydroascorbate reductase, may be among the components that regulate Golgi apparatus secretion.

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca 2+ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca 2+ -specific ionophore, Ca 2+ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.

Summary Intact cells are known to reduce external, impermeable electron acceptors. We now show that cells can reduce the iron in diferric transferrin at the cell surface and that this reduction reaction depends on the transferrin receptor as well as the transmembrane electron transport system. Reduction of external diferric transferrin is accompanied by oxidation of internal NADH which indicates that the transmembrane enzyme is an NADH diferric transferrin reductase. Highly purified liver plasma membranes have NADH diferric transferrin reductase activity which shows properties similar to the diferric transferrin reductase activity of intact cells. Cell growth stimulation by diferric transferrin and other impermeable oxidants which can react with the diferric transferrin reductase can be based on electron transport through he plasma membrane.

A fifteen minute incubation of spinach chloroplasts with the divalent Ca 2+ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn 2+ site, and that the first Ca 2+ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn 2+ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca 2+ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca 2+ site II activity can be restored with 20 mM CaCl 2 . Ca 2+ site I responds to Ca 2+ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca 2+ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF 1 , the coupling factor, from chloroplast membranes, and since Mg 2+ ions are ineffective in restoring activity, it is concluded that Ca 2+ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.