Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.     

The effects of bovine serum albumin and oleic acid on rat pancreatic lipase and bovine milk lipoprotein lipase

The effects of bovine serum albumin on rat pancreatic lipase and bovine milk lipoprotein lipase were studied in a system of triacylglycerol emulsions stabilized by 1 1 mg/ml albumin. At concentrations greater than 1 mg/ml, albumin inhibited the activity of pancreatic lipase and interfered with enzyme binding to emulsified triacylglycerol particles. These effects could be countered by occupying five fatty acid binding sites on albumin with oleic acid. Following an initial lag period which increased with albumin concentrations, enzyme activity escaped from inhibition presumably due to saturation of fatty acid sites on albumin with oleic acid. Pancreatic lipase was active at 1 mg/ml albumin and 1 mM emulsion-bound oleic acid in the system. The effects of albumin on lipoprotein lipase were diametrically opposed to the above; enzyme activity was completely inhibited by 0.1 mM oleic acid, it increased with increasing fatty acid-free albumin concentrations and decreased as the fatty acid sites on albumin were filled. At 1 mM oleic acid and no added albumin the enzyme failed to bind at the oil water interface, whereas fatty acid-free or saturated albumin had no effect on binding. It is concluded that if the inhibition of pancreatic lipase by albumin is due to the inaccessibility of the enzyme to an oil-water interface blocked by denatured albumin, then albumin saturated with oleic acid would seem to be protected from unfolding at the interface and more readily displaced by the lipase. Pancreatic lipase and lipoprotein lipase, although sharing a number of common features, are distinct enzymes both functionally and mechanistically.     

Solubilization of the alternative oxidase of cuckoo-pint (Arum maculatum) mitochondria. Stimulation by high concentrations of ions and effects of specific inhibitors.

Selective solubilization of cyanide- and antimycin-insensitive duroquinol oxidase activity from cuckoo-pint (Arum maculatum) mitochondria was achieved using taurocholate. Inhibitor-sensitivities and water-forming DQH2 (tetramethyl-p-hydroquinone, reduced form): O2 stoichiometry were the same for the alternative oxidase of intact Arum mitochondria. Cyanide-insensitive oxidation of DQH2 by intact and solubilized mitochondria was stimulated by up to four-fold by high concentrations of anions high in the Hofmeister series, such as phosphate, sulphate or citrate. Optimal (0.7 M) sodium citrate increased Vmax. for DQH2 oxidation by the solubilized preparation from 450 to 2400 nmol of O2 X min-1 X mg of protein-1 and decreased the apparent Km for DQH2 from 0.53 to 0.38 mM. Inhibition of solubilized DQH2 oxidase activity by CLAM (m-chlorobenzhydroxamic acid) and SHAM (salicylhydroxamic acid) was mixed competitive/non-competitive, with apparent inhibition constants for CLAM of 25 microM (Ki) and 81 microM (KI) and for SHAM of 53 microM (Ki) and 490 microM (KI). Propyl gallate and UHDBT were non-competitive inhibitors with respect to DQH2 (apparent Ki = 0.3 microM and 12 nM respectively). Low concentrations of C18 fatty acids selectively inhibited cyanide-insensitive oxidation by intact and solubilized mitochondria, and inhibition was reversed by 1% (w/v) bovine serum albumin. Inhibition was competitive with DQH2, suggesting that fatty acids interfere reversably with the binding of DQH2 to the oxidase. These results tend to support the view that quinol oxidation by the alternative pathway of Arum maculatum mitochondria is catalysed by a quinol oxidase protein, rather than by a non-enzymic mechanism involving fatty acid peroxidative reaction. [Rustin, Dupont & Lance (1983) Trends Biochem. Sci. 8, 155-157; (1983) Arch. Biochem. Biophys. 225, 630-639].     

Activation and transport of fatty acids in ovarian mitochondria: effect of Lh

Enzymes of fatty acid activation and transport were studied in luteinized rat ovaries. Luteal mitochondria were found to contain high levels of palmitoyl-CoA synthetase and carnitine palmitoyl-transferase activities. In addition, studies on the effect of palmitate concentration on palmitoyl-CoA synthetase activity revealed the possible existence of two forms of the enzyme: Km values of 0.34 mM and 21.33 mM, with Vmax of 3.64 and 66.67 nmoles/min/mg mitochondrial protein respectively, were obtained for the two activities. Similar kinetic data for carnitine palmitoyl-transferase activity in intact mitochondria are a Km of 21 microM and a Vmax of 18.2 nmoles/min/mg mitochondrial protein. Only one activity of this enzyme could be detected in luteal mitochondria. It appears that the activities of both enzymes were not affected by prior administration of LH in vivo. The possibility that this negative finding was due to the experimental procedures employed, rather than a reflection of the situation in vivo, could not be discounted, although its more likely that these two enzymes are probably not locus of LH stimulation. The results indicate that fatty acid oxidation is an important metabolic capability of luteal mitochondria, and support the view regarding the lipid nature of the respiratory fuel of ovarian tissue.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Hepatic lipase. Purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200 000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53 000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55 000, 66 000 and 2600 mumol fatty acid/mg per h with apparent Michaelis constant (Km) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotein C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.     

Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria.

1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.     

Characterization of lipases from the lipid bodies and microsomal membranes of erucic acid-free oilseed-rape (Brassica napus) cotyledons.

Lipase (triacylglycerol lipase, EC 3.1.1.3) activities have been reported previously in the lipid body and microsomal membranes of oilseed-rape (Brassica napus cv. Andor) seedlings, but conflicting data made it unclear whether there was one lipase in the lipid bodies, with the microsomal activity being attributable to fragments of lipid-body membrane, or if there were two separate lipase activities. In the present study, simultaneous characterization of the lipases under identical conditions showed they differed substantially in their pH-activity curves, kinetics and substrate specificities. (1) The kinetics of the microsomal lipase showed that the rate of lipolysis reached a plateau at concentrations above 5 mM, whereas the lipid-body lipase showed a linear increase in activity with substrate concentration up to 20 mM. (2) The pH optimum of the microsomal lipase was 7.5, whereas that of the lipid-body lipase was 9.0. The microsomal lipase was greatly inhibited at higher pH values, whereas the lipid-body lipase was much less affected. (3) Activity of the microsomal lipase was greatly diminished when substrates with longer chain length were used, and enhanced 4-fold if the substrates contained a single double bond. The lipid-body lipase was relatively unaffected by the type of fatty acid in the triacylglycerol. (4) SDS/polyacrylamide-gel electrophoresis showed little or no cross-contamination of the lipid-body and microsomal fractions. (5) The microsomal lipase activity comprised 75-80% of the total extracted.     

Partial purification and properties of rat-heart adenosine kinase.

The activity of myocardial adenosine kinase (E.N. 2.7.1.20) in a number of species was assayed. Rat heart contained the highest specific activity. From this source adenosine kinase was purified in a simple way 80-fold, until it was free of adenosine deaminase activity. A molecular weight of about 39 000 was measured. NSC 113939 (1), NSC 113940 and 8-azaadenosine inhibited myocardial adenosine kinase. Dipyridamole stimulated the enzyme at high adenosine levels, and inhibited at low substrate concentrations. A number of divalent cations could (partially) substitute for Mg2+. The optimal concentration of MgCl2 or MnCl2 was about 0.5 mM; concentrations exceeding 1 mM inhibited severely. An apparent Km for ATP of 0.1 mM was measured, whereas an apparent Km for adenosine of 0.5 muM was was found. The latter increased to 3.3 muM, when dipyridamole was added. Replacement of ATP by GTB or ITP increased the activity, and UTP and CTP were inferior as a phosphate donor.     

The aromatization of cyclohexanecarboxyl-CoA to hippuric acid by guinea pig liver mitochondria: submitochondrial localization

Summary The conversion of cyclohexanecarboxyl-CoA to hippuric acid in submit ochondrial fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxyl-CoA to hippuric acid converting system (CCoAHC-system) and marker enzymes in the various submit ochondrial fractions showed that the CCoAHC-system is localized in the mitochondrial matrix. Partial separation of the inner and outer membranes has been accomplished by treating mitochondria with digitonin in isotonic medium and fractionating the treated mitochondria by differential centrifugation. A digitonin-protein ratio of 2.6 mg of digitonin/10 mg of protein must be used in order to release significant amounts of amine oxidase activity (outer membrane marker) from low speed mitochondrial pellets. This pellet still contained most of the glutamate dehydrogenase activity and was insignificantly contaminated with adenylate kinase. Moderate concentrations of phenazine methosulfate (PMS) greatly stimulated the activity of the CCoAHC-system, even in intact mitochondria (optimal concentration of PMS: 1 mM) whilst higher concentrations (> 1 mM) decreased the activity. The formation of hippuric acid in these mitochondrial preparations was linear with time for at least 40 min and linear with respect to protein concentration up to approximately 2.0 mg mitochondrial protein·m¹.     

An early steroidogenic defect in hormone-induced Leydig cell desensitization

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.     

Triacylglycerol lipase activity in the rabbit renal medulla

Although the renal medulla is rich in triacylglycerols, the lipolysis of these intracellular triacylglycerols by a renomedullary triacylglycerol lipase has not been directly demonstrated. The present study demonstrates triacylglycerol lipase activity localized in the particulate subcellular fractions of rabbit renal medullae. Renomedullary triacylglycerol lipase activity, as determined by the hydrolysis of [14C]triolein to [14C]oleic acid, was observed to have a pH optimum of 5.8. Addition of cAMP/ATP/magnesium acetate resulted in an 80% activation of crude homogenate triacylglycerol lipase activity; addition of exogenous cAMP-dependent protein kinase resulted in a further activation of lipolysis. 3 mM CaCl2 had no effect on basal triacylglycerol lipase activity. 1 M NaCl did not inhibit lipolysis, suggesting that the lipase activity measured was not due to lipoprotein lipase. Endogenous renomedullary triacylglycerols were hydrolysed by a lipase in the 100,000 X g pellet of renomedullary homogenates, resulting in the release of free fatty acids including arachidonic and adrenic acids. Dispersed renomedullary cells were prepared to monitor hormone-sensitive triacylglycerol lipase activity in intact cells. Addition of 10 microM forskolin and 10 microM epinephrine resulted in 8-fold and 50-fold increases in triacylglycerol lipase activity, respectively, as defined by release of free glycerol from the cells. These studies demonstrate that a cAMP-dependent hormone-sensitive triacylglycerol lipase is present in the renal medulla, and is responsible for the hydrolysis of renomedullary triacylglycerols.     

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship.

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58-69% recovered in the 100 000 X g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 +/- 13.6% (S.D.) and triacylglycerol lipase 11.9 +/- 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, The hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent Km for substrate and molecular weight 3-10(5)-6-10(5) by gel filtration and a minor component of lower apparent Km and heterogeneous molecular weight above 1 million, which was found mostly in complex and lipid.     

Characterization of Rat Testes Mitochondrial Retinoylating System and Its Partial Purification

Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.     

Possible role of lipoprotein lipase in the regulation of endogenous triacylglycerols in the rat heart.

1. Adrenaline has a biphasic effect on intracellular lipoprotein lipase activity and on endogenous triacylglycerol content in heparin-perfused heart. 2. A high concentration of adrenaline (1 microM in the perfusion buffer) activated endogenous lipoprotein lipase activity and, at the same time, decreased intracellular triacylglycerol stores. 3. In contrast, a low concentration (0.005 microM-adrenaline) inhibited intracellular lipoprotein lipase activity. Under these conditions, cardiac triacylglycerol content was elevated above control values. 4. Perfusing the heart with high and low concentrations of 3-isobutyl-1-methylxanthine elicited a biphasic effect on endogenous lipoprotein lipase activity and triacylglycerol content similar to that seen with adrenaline treatment. 5. The effect of adrenaline on intracellular lipoprotein lipase activity appears to be mediated by cyclic AMP through protein kinase. 6. A possible role for intracellular lipoprotein lipase in the regulation of endogenous triacylglycerol in rat heart is proposed.     

Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm

In this study we examined the processes by which malate and pyruvate are taken up across the leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.     

Palmityl-CoA and palmitylcarnitine interactions in mitochondrial fatty acid elongation

We have investigated interactions of palmityl-CoA and l-palmitylcarnitine as substrates for mitochondrial fatty acid elongation. l-Palmitylcarnitine is a more effective substrate primer for fatty acid elongation by intact mitochondria than is palmityl-CoA. Exogenous l-carnitine inhibited l-palmitylcarnitine-supported mitochondrial fatty acid elongation by both sonically disrupted and intact heart mitochondria, probably by shifting the equilibrium between palmitylcarnitine and palmityl-CoA toward palmitylcarnitine, thus removing palmityl-CoA from the reaction. d-Carnitine was without effect. d-Palmitylcarnitine inhibition of palmitylcarnitine transferase activity decreased palmitylcarnitine-stimulated mitochondrial fatty acid elongation but increased palmityl-CoA supported fatty acid elongation, presumably by increasing the effective concentration of palmityl-CoA in the assay medium. The data indicate that, although l-palmitylcarnitine is an effective substrate primer for mitochondrial fatty acid elongation, palmityl-CoA rather than palmitylcarnitine is the immediate precursor for fatty acid chain elongation.     

Reversible effects of fatty acids on respiration, oxidative phosphorylation, and heat production of rat liver mitochondria.

1. Oleic acid at low concentrations (0--70 nmol/mg protein) stimulated mitochondrial state 4 respiration 4-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -112 to -208 kJ/O and completely inhibited ATP synthesis without significant effect on the Mg-ATPase activity of mitochondria. 2. Similar effects on mitochondrial respiratory activities were observed with other fatty acids. 3. Bovine serum albumin (BSA) protected mitochondria from the effects of oleic acid irrespective of the order of addition of oleic acid and BSA to mitochondria. The capacity of BSA to bind oleic acid was calculated to be 3.6--7.1 (mean, 4.9) mol of oleic acid/mol of BSA. 4. The response time of mitochondrial respiration to added oleic acid or BSA was 20--25 s.     

Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase

The concerted action of purified bovine gastric lipase and human pancreatic colipase-dependent lipase and colipase, or crude human pancreatic juice, in the digestion of human milk triacylglycerols was explored in vitro. Gastric lipase hydrolyzed milk triacylglycerol with an initially high rate but became severely inhibited already at low concentration of released fatty acid. In contrast, colipase-dependent lipase could not, by itself, hydrolyze milk triacylglycerol. However, a short preincubation of milk with gastric lipase, resulting in a limited lipolysis, made the milk fat triacylglycerol available for an immediate and rapid hydrolysis by pancreatic juice, and also for purified colipase-dependent lipase, provided colipase and bile salts were present. The same effect was obtained when incubation with gastric lipase was replaced by addition of long-chain fatty acid. Long-chain fatty acid increased the binding of colipase-dependent lipase to the milk fat globule. Binding was efficient only in the presence of both fatty acid and colipase. We conclude that a limited gastric lipolysis of human milk triacylglycerol, resulting in a release of a low concentration of long-chain fatty acids, is of major importance for the subsequent hydrolysis by colipase-dependent lipase in the duodenum.     

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.