Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae

The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H⁺). The buffering action of passively excreted metabolites (CO₂, organic acids) and cell surface polyelectrolytes contributed only 15–40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 mmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence $\text{K}{}^{\mathrm{+}}\text{>}\text{Rb}{}^{\mathrm{+}}\text{>>}\text{Li}{}^{\mathrm{+}}\text{～-}\text{Cs}{}^{\mathrm{+}}\text{～-}\text{Na}{}^{\mathrm{+}}$. The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO₂ and organic acids, net extrusion of H⁺ and K⁺ (in K⁺-free media; in K⁺-containing media this is preceded by an initial rapid K⁺ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

Difference in susceptibility of arginine-vasopressin and oxytocin to aminopeptidase activity in brain synaptic membranes

Arginine-vasopressin and oxytocin, peptides which serve as putative precursors for neurotrophic fragments, were digested in the presence of the respective ¹⁴C-Tyr²- and ${}^{14}\text{C-GlyNH}{}_2{}^9\text{-labeled}$ nonapeptides with a purified synaptic membrane preparation of rat brain. In this preparation aminopeptidase activity predominates in the conversion of these peptides. The disappearance of intact peptide and the release of free ¹⁴C-Tyr and ¹⁴C-GlyNH₂ was followed simultaneously with time by HPLC. Oxytocin was about four times more resistant to proteolysis than arginine-vasopressin as measured by slower disappearance of intact oxytocin, and reflected by the slower release of ¹⁴C-Tyr, but not of ¹⁴C-GlyNH₂ from oxytocin. Comparison of degradation rates of structure analogues showed that peptides having Ile in position 3, as oxytocin, were more resistant than analogues having Phe in position 3, as arginine-vasopressin. The data demonstrate that arginine-vasopressin and oxytocin differ markedly in susceptibility to the aminopeptidase activity in brain synaptic membranes, and indicate that this difference resides primarily in the amino acid residue in position 3. It is suggested that the difference in susceptibility may affect the pattern of neurotrophic metabolites in brain.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids.

The dual wavelength assay technique (H. R. Levy, and G. H. Daouk, 1979, J. Biol. Chem.254, 4843–4847) is used to examine the rates of the NADP- and NAD-linked reactions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase simultaneously under various conditions. Inhibition by ATP, MgATP^2?, acetyl-CoA, and palmitoyl-CoA is greatly diminished at high glucose 6-P concentration which favors the NAD-linked reaction. Increasing $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction. The selective effects of glucose 6-P and the $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratio, which cannot be detected by conventional assays, are explained in terms of the differing kinetic mechanisms for the NADP-linked and NAD-linked reactions previously described (C. Olive, M. E. Geroch, and H. R. Levy, 1971, J. Biol. Chem.246, 2047–2057). It is proposed that these effects constitute the mechanism whereby the nucleotide specificity of the amphibolic glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides is regulated.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Metabolic effects of glucose deprivation and of various sugars in normal and transformed fibrobalst cell lines.

The sugar composition of the growth medium influenced the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, pyruvate and lactate production, and ATP levels in both normal and transformed fibroblast cell lines growing in tissue culture. Removal of glucose led to a rapid three- to fourfold rise in the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, followed by a slower decline in the content of ATP. However, there was no change in the adenylate energy charge [$\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP + ADP + AMP}\text{)}$] over a 2-h period. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio was restored to the original level within 10 s of glucose readdition. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ratios in cell lines growing on galactose were as high as for those incubated without sugars; growth on mannose or fructose produced intermediate ratios. There was an inverse relationship between the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio and pyruvate-lactate production for glucose, fructose and galactose. Thus, all cell lines had a high production of pyruvate and lactate but a low $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio when grown on glucose. In contrast, when galactose served as the sugar source, acid production was low, while the ratio was high. All cell lines had comparable hexokinase activity, and glucose was the best substrate, mannose intermediate and fructose poorest. Hexokinase activity did not correlate with the relative degree of utilization of the sugars. These results suggest that the sugar composition of the growth medium affects the metabolic pattern of a cell line, including the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, the ATP content and the production of pyruvate and lactate.     

Female dimorphism in honey-bee larvae with respect to glucose metabolism

The rate of conversion of glucose-1-¹⁴C and glucose-6-¹⁴C to ¹⁴CO₂ and lipid was monitored in queen and worker larvae between 48 and 96 hr of age. A definite dimorphism in glucose metabolism between castes was established. Worker larvae 72 hr of age have a much greater $\text{C}{}_6\text{C}{}_1$ ratio than do queen larvae of the same age. The ratios were of the same order of magnitude for both castes of larvae younger or older than 72 hr. Queen larvae were shown to have a greater rate of lipid synthesis than worker larvae.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

The stimulating effect of 3′,5′-(cyclic)adenosine monophosphate and lipolytic hormones on 3-O-methylglucose transport and 45Ca2+ release in adipocytes and skeletal muscle of the rat

(1) In order to assess the possible role of 3′,5′-(cyclic)adenosine monophosphate (cAMP) in the control of glucose transport, the effect of the nucleotide or agents known to increase its intracellular concentration on sugar transport or ⁴⁵Ca²⁺ washout were characterized in epididymal fat pads, free fat cells and soleus muscles of the rat. (2) When added to the incubation medium, cAMP (0.1–2.0 mM) stimulated $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ washout from fat pads. This effect was abolished by cytochalasin B, and additive to that induced by submaximal (10–25 μU/ml), but not by supramaximal (10 mU/ml) concentrations of insulin. (3) cAMP (2 mM) stimulated the conversion of [U-¹⁴C]glucose into CO₂ and triacylglycerols. This effect was additive to that of insulin (100 μU/ml). (4) ACTH, glucagon, adrenaline, noradrenaline and salbutamol, which are all known to increase the cAMP content of adipose tissue, stimulated the washout of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca²⁺ from preloaded fat pads. The fractional losses of the two isotopes were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.73}$). (5) In free fat cells, adrenaline (10^?6 M) and salbutamol (10^?5 M) stimulated the uptake of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$, and salbutamol (10^?5 M) did not interfere with the stimulating effect of insulin (25 μU/ml) on sugar uptake. (6) In rat soleus muscles, adrenaline and salbutamol produced a dose-dependent stimulation of the washout of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca²⁺. The effect of adrenaline on sugar efflux was abolished by propranolol. (7) It is concluded that the activation of the glucose transport system by insulin is unlikely to be mediated by a drop in the cellular concentration of cAMP. An increase in cAMP brought about by β-adrenoceptor agonists or lipolytic hormones may induce a mobilization of calcium ions from cellular pools into the cytoplasm, which in turn leads to the activation of the glucose transport system demonstrated in the present as well as in several earlier studies.     

Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ${}^{14}\text{C}{}^3\text{H}$ ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ${}^{14}\text{C}{}^3\text{H}$ incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane

Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimidelabeled}$ mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 10³. The nmol $\text{N-[}{}^3\text{H}\text{]-}\text{ethylmaleimide}$ bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide/mol}$ cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$ and identified five labeled proteins. Only the labeled 32 · 10³ dalton and the 45 · 10³ dalton proteins are common to both systems