Activation of glutamate dehydrogenase by L-leucine

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.     

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations.

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.     

Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase.

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

Activation of Alpha Chymotrypsin by Three Phase Partitioning Is Accompanied by Aggregation

Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242–1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130–462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in λ_max of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.     

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol.

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 degrees C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.     

Effects of proteinase inhibitors on adenylate cyclase.

The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5'-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05--0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25--50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5'-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10--20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.     

Changes of Acetylcholinesterase Activity in Brain Areas and Liver of Sucrose- and Ethanol-Fed Rats

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50°C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30°C (control or sucrose groups) or 34–35°C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250–500 mM), while at lower concentrations (10–25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consecuently the behaviour of acetylcholinesterase.     

Perinatal developmental changes in hepatic UDP-glucuronyltransferase.

Postnatal developmental changes in hapatic microsomal UDP-glucuronyltransferase were studied in the rat. The previously reported postnatal decline in the capacity of microsomal fractions to glucuronidate p-nitrophenol was found to be observable in unperturbed preparations only at non-saturating concentrations of the substrate UDP-glucuronic acid. At saturating concentrations of UDP-glucuronic acid, activity is identical in newborns and adults. Kinetic analysis revealed that the enzyme from liver of newborns has a much higher affinity for UDP-glucuronic acid than does the enzyme in adults, but the same activity at Vmax. On the other hand, the enzyme from adult liver microsomal fractions can be activated by the physiological allosteric effector UDP-N-acetylglucosamine, whereas the enzyme from newborns is largely unaffected by it. Thus it appears that the number of enzyme active sites is not changing; rather, the enzyme is maturing to a more highly regulable form. There were also differences between the enzymes in newborns and adults in their response to perturbation of the membrane-lipid environment by detergent and phospholipase A. Possible interpretations of these differences are discussed.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.     

Effect of enhanced red blood cell aggregation on blood flow resistance in an isolated-perfused guinea pig heart preparation

The role of red blood cell (RBC) aggregation as a determinant of in vivo blood flow is still unclear. This study was designed to investigate the influence of a well-controlled enhancement of RBC aggregation on blood flow resistance in an isolated-perfused heart preparation. Guinea pig hearts were perfused through a catheter inserted into the root of the aorta using a pressure servo-controlled pump system that maintained perfusion pressures of 30 to 100 mmHg. The hearts were beating at their intrinsic rates and pumping against the perfusion pressure. RBC aggregation was increased by Pluronic (F98) coating of RBC at a concentration 0.025 mg/ml, corresponding to about a 100% increment in RBC aggregation as measured by erythrocyte sedimentation rate. Isolated heart preparations were perfused with 0.40 l/l hematocrit unmodified guinea pig blood and with Pluronic-coated RBC suspensions in autologous plasma. At high perfusion pressures there were no significant differences between the flow resistance values for the two perfusates, with differences in flow resistance only becoming significant at lower perfusion pressures. These results can be interpreted to reflect the shear dependence of RBC aggregation: higher shear forces associated with higher perfusion pressures should have dispersed RBC aggregates resulting in blood flow resistances similar to control values. Experiments repeated in preparations in which the smooth muscle tone was inhibited by pre-treatment with papaverine indicated that significant effects of enhanced RBC aggregation could be detected at higher perfusion pressures, underlining the compensatory role of vasomotor control mechanisms.     

Inhibition of cyclooxygenase activity and platelet aggregation by epoxyeicosatrienoic acids. Influence of stereochemistry

Certain epoxyeicosatrienoic acids (EETs) that were not cyclooxygenase substrates were effective cyclooxygenase inhibitors. Both (+/-)-14,15-cis-EET and (+/-)-8,9-cis-EET inhibited purified enzyme at concentrations from 1 to 50 microM; (+/-)-11,12-cis-EET was ineffective at concentrations below 100 microM. For the case of 14,15-cis-EET, only the (14R,15S)-stereoisomer was active. Other isomers including (14S,15R)-cis-EET, (14R,15R)-trans-EET, (14S,15S)-trans-EET, and the erythro and threo vicinal 14,15-diols were inactive. In addition to their effects on isolated enzyme preparations, cyclooxygenase activity in platelet suspensions, reflected by thromboxane B2 formation, was also inhibited by (14R,15S)-cis-EET and (+/-)-8,9-cis-EET but not by the other isomers. Thus potency and stereospecificity requirements were maintained for cyclooxygenase within intact platelets. Unlike the stereospecific inhibition of the cyclooxygenase enzyme, platelet aggregation induced by arachidonic acid was inhibited by all EET isomers at concentrations from 1 to 10 microM with no evident stereospecificity. Inhibition of aggregation was not uniformly associated with inhibition of thromboxane B2 formation; ordinarily, these two parameters correlate closely. This dissociation was not maintained for another biochemical process involved in platelet activation. For instance, there was a uniform correlation between inhibition of phosphorylation of a 40-kDa platelet protein and inhibition of aggregation. Our results suggest that effects of EET may originate from either stereospecific or nonspecific mechanisms. Definition of such mechanisms may be important to appreciate any physiological relevance of these substances.     

Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-acetyl-alpha-galactosaminyl-R beta 3-galactosyltransferase

Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAc alpha-R beta 3-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed that uncharged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of beta 3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moieties in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the beta 3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain beta 3- and beta 6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAc beta 3GalNAc alpha-R) and core 4 [GlcNAc beta 6(GlcNAc beta 3)GalNAc alpha-R]. These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with beta 3-Gal-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of homogeneous preparations of hog kidney myo-inositol oxygenase by quinolinic acid and ferrous ions

When incubated and assayed with 0.6 mM quinolinate and 1.0 μM Fe(II), some homogeneous preparations of hog kidney myo-inositol oxygenase (EC 1.13.99.1) which have been stored at ?20° for weeks to months have a catalytic activity which is 4 to 5 fold higher than found under any other conditions. This high activation is very dependent on activator concentrations and temperature, and is not observed with freshly prepared (never frozen) preparations of homogeneous oxygenase. A polymer of the enzyme formed on storage is believed to be responsible for the high activities. 3-Mercaptopicolinate is also an effective activator under similar conditions but 3-amino-picolinate is not. The similar but opposite effects of these compounds on phosphoenolpyruvate carboxykinase are noted, and the possible relevance of these findings to the control of the oxygenase in vivo is considered.     

The dependence on vitamin E and selenium of drug demethylation in rat liver microsomal fractions.

1. The effects of vitamin E deficiency, and of vitamin E and selenium deficiency, on rat liver microsomal aminopyrine demethylase activity were investigated. It was found that, over a wide range of substrate concentrations, the enzyme activity in preparations from deficient animals was significantly lower than that in controls. 2. Addition of antioxidants in vitro, either to the homogenization or to the assay media, was without significant effect on the depressed enzyme activity. Castration and alteration in dietary protein concentration were also without effect. The rate of oxidation of NADPH was however, lower in preparations from deficient animals. 3. Lineweaver-Burk plots of the reciprocal of enzyme activity and substrate concentration showed a higher Km value in preparations from vitamin E-deficient animals, irrespective of whether selenium was present; the Vmax. was unaffected. These parameters were unchanged when antioxidants were added in vitro. Induction with phenobarbitone and 3-methylcholanthrene showed large changes in Km value which, for preparations from vitamin E-deficient animals, was higher than that for corresponding controls. 4. Examination of the synergism between NADH and NADPH as donors of reducing equivalents for aminopyrine demethylation showed that vitamin E and selenium were only minimally involved in the phenomenon. However, both the initial rate and the extent of demethylation were significantly lower in vitamin E- and selenium-deficient preparations and both nutrients were required for the restoration of full activity. 5. The significance of these results is discussed in the light of our working hypothesis.     

The stimulation of liver phosphorylase b by AMP, fluoride and sulfate. A technical note on the specific determination of the a and b forms of liver glycogen phosphorylase.

1. The activity of liver phosphorylase b from several mammalian species has been studied. The enzyme from rat or mouse has a higher activity than the rabbit enzyme, which is itself more active than pig liver phosphorylase b. 2 The activity of liver phosphorylase b is influenced by anions and by AMP, and these effects are influenced by pH. Fluoride, which is currently added to the assay mixture of phosphorylase a in crude preparations, is about as active as sulfate as a stimulator of phosphorylase b. 3. When assayed at pH 6.1 and in the presence of 0.15 M NaF, the activity of rat liver phosphorylase b reaches 25% of that of the a enzyme; if 1 mM AMP is also present, this value rises to 50%. 4. Methods are described that allow the determination of liver phosphorylase a without interference of b, and the determination of total phosphorylase (a+b) in rat liver.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

The effect of soluble rat liver proteins on the activity of microsomal stearoyl-CoA and linoleoyl-CoA desaturase.

1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.