Low Citrate Concentrations Research Articles

Interaction of citrate with Fe(III)-bleomycin

1. 1. Citrate binds to Fe(III)-bleomycin, removing the ferric ion from the iron-drug complex; a reaction that may be of physiological significance. 2. 2. Low concentrations of citrate markedly enhance the rate of iron transfer from Fe(III)-bleomycin to apotransferrin; an iron binding plasma protein.

An 'in situ' perfusion system suitable for investigating mammary-tissue metabolism in the lactating rat. Hormonal regulation of acetyl-CoA carboxylase.

A technique is described for the non-recirculating perfusion of inguinal/abdominal mammary tissue in situ in anaesthetized lactating rats. Tissue viability was maintained, without resort to infusion of vasoactive chemicals which may also be effectors of cellular metabolism, for at least 90 min. Total tissue adenine nucleotides (per mg of DNA) were somewhat decreased in perfused relative to non-perfused mammary tissue. DNA content (per g wet wt. of tissue) was diminished after 90 min of perfusion to approx. 65% of its value in control tissue. Adenylate energy-charge ratios were lower in perfused tissue in the absence of hormones than in control tissue. They were increased to control values by the presence of either insulin or isoprenaline in the perfusate. No changes occurred in flow rate of the perfusate that might account for these increases. In mammary tissue perfused without addition of hormones, acetyl-CoA carboxylase activities were similar to those measured in control tissue samples, although activity-ratio measurements implied some increase in the phosphorylation of this enzyme. Insulin or isoprenaline increased the activity of acetyl-CoA carboxylase, especially when this was measured at low concentrations of citrate. Confirming conclusions from previous experiments with mammary acini and explant preparations, insulin activated acetyl-CoA carboxylase in mammary tissue, but inhibition of its activity was not mediated by cyclic AMP.

Retention of Iron by Rat Intestine in Vivo as Affected by Dietary Fiber, Ascorbate and Citrate

The effects of pH, ascorbate, citrate and dietary fiber on retention of ferrous and ferric iron by jejuno-ileal segments of rat intestine were examined in vivo. Iron was introduced in an isosmotic solution of sodium chloride and dextrose buffered by 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES) and acetate. Stabilization of the iron solutions was aided by use of iron concentrations ≤ 1 µg/ml injected into the intestine for 10-min periods. Iron retention was optimal over a broad pH range from 5 to 7.8. Inclusion of ascorbic acid in the solution injected (5, 25 or 75 µg/ml) did not increase retention of iron in either valence state. A low concentration of sodium citrate (2 mM) had no effect on iron retention, but increasing the concentration to 5 mM released iron from the mucosa. Maize and wheat fibers decreased the retention of ferrous iron by binding and by promoting autoxidation and formation of poorly soluble iron polymers. Bound ferrous iron was released completely at pH below 5. Retention of ferric iron was also lowered in the presence of fiber, presumably as a result of polymerization. Retention of iron by the rat in the absence of ligands was independent of valence state.

The effect of citrate, calcium, and magnesium ions on the potassium movement across the human platelet membrane.

The intracellular K+ concentration in platelets is reduced during storage in citrated plasma. As shown previously, pH has a marked influence on this reduction. The effect of citrate and Ca2+ and Mg2+ on the K+ permeability of the membrane and on the active K+ transport across the membrane was investigated. Platelets were incubated in dialyzed plasma at 37 degrees C, pH 5.5 to 7.9, with and without added Ca2+ and Mg2+ and citrate. After 60- to 120-minute incubation, a steady state was reached, and ouabain was added to inhibit the active K+ transport. In the pH interval 5.5 to 6.2 citrate, Ca2+ and Mg2+ had no influence on either the active K+ transport or the K+ permeability. Between pH 7.1 and 7.9, Ca2+ and Mg2+ decreased the permeability, whereas citrate increased it. At pH 7.9, citrate had a depressing effect on the active transport. These results indicate that a low citrate concentration and a pH below 7 are important for maintaining a high intracellular K+ of platelets during storage in plasma.

Citrate activation of NAD-specific isocitrate dehydrogenase from bovine heart.

NAD-dependent isocitrate dehydrogenase from bovine heart was activated by lower concentrations of citrate in the presence of ADP (apparent S0.5 citrate = 0.033 mM) than in its absence (apparent S0.5 citrate = 2.64 mM) at low magnesium DL-isocitrate (0.18 mM) and free DL-isocitrate3- (0.45 mM) concentrations. Under these conditions, citrate (0.3 mM) lowered the apparent S0.5 for ADP from 0.24 to 0.05 mM. The binding of NAD+ was unaffected by citrate; however, saturating concentrations of citrate lowered the apparent S0.5 for magnesium isocitrate from 0.63 to 0.19 mM in the presence of ADP. Citrate does not appear to bind to the regulatory or catalytic magnesium isocitrate-binding sites, since the Hill coefficient for magnesium isocitrate was not lowered by citrate nor did inhibition occur at high citrate concentrations. The data suggest that magnesium citrate was the activating species. Citrate activation occurred from pH 6.5 to 8.0. As with magnesium citrate, calcium citrate lowered the S0.5 for magnesium isocitrate (apparent S0.5 for calcium citrate = 0.26 mM), and it did not appear to bind to the regulatory or catalytic magnesium isocitrate-binding sites. However, in contrast to the substantial facilitation of magnesium citrate activation by ADP, no activation by calcium citrate occurred in the presence of ADP. Differences in the mechanism of activation of the enzyme by magnesium citrate and calcium citrate were also indicated by the finding that, whereas a number of tricarboxylates could replace citrate as an activator with Mg2+ as the sole divalent cation activator, only citrate was effective for the further enhancement of velocity by added calcium.

Partial Purification and Properties of an Alkaline α-Galactosidase from Mature Leaves of Cucurbita pepo

A fourth molecular from of alpha-galactosidase, designated L(IV), an alkaline alpha-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-alpha-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K(m) determinations in McIlvaine buffer (200 millimolar Na(2)-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-alpha-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L(IV) was partially inhibited by Ca(2+), Mg(2+), and Mn(2+), more so by Ni(2+), Zn(2+), and Co(2+), and highly so by Cu(2+), Ag(2+), Hg(2+) and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-alpha-d-galactoside or by myo-inositol, but alpha-d-galactose was a strong inhibitor. As observed for most other forms of alpha-galactosidase, L(IV) only catalyzed the hydrolysis of glycosides possessing the alpha-d-galactose configuration at C(1), C(2), and C(4), and did not hydrolyze p-nitrophenyl-alpha-d-fucoside (alpha-d-galactose substituted at C(6)). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-alpha-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L(IV) in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L(IV)in vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L(IV) activity through coordination with some toxic cation introduced as a buffer contaminant.

Regulation of primary alkylsulfatase induction in Pseudomonas C12B: concentration-dependent stimulation-inhibition by exogenous UTP and sodium acetate and inhibition by 1-hexanol.

Pseudomonas C12B, an isolate from detergent-enriched soil, synthesized primary alkylsulfatase in response to sodium hexan-1-yl sulfate as the inducer. The induction of this enzyme was inhibited by exogenous 1 mM UTP but not by ATP or other nucleoside triphosphates. The uridine nucleotide was about 10-fold more effective than other uracil-related effectors and the ability of the nucleotide (0.1-1.0 mM) to inhibit induction was dependent upon the presence of added Mg2+. At concentrations less than 0.1 mM, UTP stimulated induction and the extent of this effect was also Mg2+ dependent. Marked stimulation of induction also occurred in response to low (less than or equal to 2.5 mM) concentrations of acetate, citrate, and succinate. However, only acetate inhibited induction (by 64%) at higher (20 mM) concentrations. 1-Hexanol was a more effective inhibitor. An 80% reduction in activity was recorded after exposure of cells to 5 mM 1-hexanol. At this concentration, 2-hexanol was without effect and 3-hexanol inhibited induction by 35%. Simultaneous exposure of the cells to 2,4-dinitrophenol failed to reverse hexanol- or acetate-mediated inhibition. It is suggested that 1-hexanol per se regulates alkylsylfatase induction and immediate product inhibition was proposed as a term to describe this type of inhibition.

Effects of acetylsalicylic acid, sulfinpyrazone and dipyridamole on platelet adhesion and aggregation in flowing native and anticoagulated blood.

The interaction of rabbit platelets with subendothelium of rabbit aorta was investigated under controlled blood flow conditions. Platelet adhesion and thrombus formation were compared after perfusion of native blood and of blood anticoagulated with citrate, heparin or heparin plus citrate. 15 mM citrate in plasma caused significant reduction of aggregation, thrombus volume and thrombus height; adhesion was concomitantly increased. Heparin (500 U/kg) had no significant effect on adhesion and thrombus dimensions. Treatment of rabbits with acetylsalicylic acid or sulfinpyrazone caused a significant reduction of thrombus volume and thrombus height in the presence of citrate. However, no significant effects were observed in native or heparinized blood. It is concluded that low citrate concentrations: (1) inhibit thrombus growth; (2) enhance thrombus breakdown; (3) therefore increase adhesion, and (4) strongly enhance a possible inhibitory effect of acetylsalicylic acid and sulfinpyrazone on thrombus growth.

Purine nucleoside diphosphate regulation of yeast hexokinases

The low activity state of hexokinase P-II, originally produced by Kosow and Rose by lowering the pH from 8 to 7 in certain sulfonated buffers, is not observed in Tris or imidazole buffers at pH 7 unless low concentrations of ADP or GDP are added. At pH values below 7 in imidazole buffer, partial inhibition occurs by protonation alone, and ADP or GDP causes further inhibition. As in the Kosow-Rose experiments, the enzyme in the low activity state can be activated either by excess ATP or by low concentrations of citrate, 3-phosphoglycerate and other metabolites. The inhibition by nucleoside diphosphates is greater at high glucose concentration. Hexokinase P-I is much less susceptible to regulation by nucleoside diphosphates or citrate, suggesting different physiological roles for the two isoenzymes.

Regulation of rat liver acetyl-CoA carboxylase. Stimulation of phosphorylation and subsequent inactivation of liver acetyl-CoA carboxylase by cyclic 3‘:5‘-monophosphate and effect on the structure of the enzyme.

The effects of citrate and cyclic AMP on the rate and degree of phosphorylation and inactivation of rat liver acetyl-CoA carboxylase were examined. High citrate concentrations (10 to 20 mM), which are generally used to stabilize and activate the enzyme, inhibit phosphorylation and inactivation of carboxylase. At lower concentrations of citrate, the rate and degree of phosphorylation are increased. Furthermore, phosphorylation and enzyme inactivation are affected by cyclic AMP under these conditions. At high citrate concentrations, cyclic AMP has little or no effect on inactivation and phosphorylation of acetyl-CoA carboxylase. Phosphorlation and inactivation of carboxylase is accompanied by depolymerization of the polymeric form of the enzyme into intermediate and protomeric forms. Depolymerization of carboxylase requires the transfer of the gamma-phosphate group from ATP to carboxylase. Inactivation occurs in the absence of CO2, which indicates that phosphorylation of the enzyme is the cause of inactivation and depolymerization, i.e. carboxylation of the enzyme is not responsible for inactivation of the enzyme.

The importance of pH and citrate concentration on the in vitro and in vivo behavior of Radiogallium

The influence of pH and citrate concentration on radio labeled gallium citrate has been investigated in vitro and in vivo as an aid to understanding the behavior of this radiopharmaceutical. Using paper chromatography, we have shown that a competition for gallium exists between hydroxyl and citrate ions in solution such that effective chelation occurs only at low pH or high citrate concentration. The influence of preparation pH has been investigated in tumour-bearing rats; the biodistribution of 67Ga was found to be virtually unaffected by pH in the range of 6–10. The influence of citrate on dog serum-protein binding has been studied using gel filtration and equilibrium dialysis; our results show that a competition for gallium exists in serum environments between serum proteins and citrate such that an increase in citrate concentration will result in dissocation of gallium bound to protein. The results of this study indicate that gallium citrate is unstable in vivo and that gallium exists in serum environments largely bound to serum proteins.

The effects of pCO 2 and pH on platelet shape change and aggregation for human and rabbit platelet-rich plasma

The rapid changes in pH, pCO 2 and pO 2 of human and rabbit platelet-rich plasma (PRP) were measured during processing and storage in air in closed tubes and cuvettes. The pH increased from 7.3 to 7.8 and pO 2 from 20 to 130 mmHg, while pCO 2 decreased from 55 to 12 mmHg, after post-bleeding times of 150 minutes. The effects of pCO 2 and pH on platelet shape change and aggregation induced by ADP, serotonin and adrenaline were studied with controlled physiologically normal pCO 2 (35 mmHg) and pH (7.4) versus low pCO 2 (15 mmHg) and high pH (7.8) conditions. At the controlled physiologically normal pCO 2 and pH conditions, rabbit PRP gave reduction in primary and secondary aggregation with serotonin, and reduction in primary aggregation with adrenaline, as compared to low pCO 2 and high pH. However, ADP-induced primary aggregation was insensitive to these pCO 2 and pH changes for human and rabbit PRP anticoagulated with heparin or with low concentration of sodium citrate, but became markedly inhibited with increasing citrate concentrations. At the normally high pCO 2 and low pH, ADP- and serotonin-induced shape change were not significantly altered. These results suggest that the pCO 2 and/or pH might be affecting the transmission process between surface receptors and final aggregation, with probable dependence on available calcium.

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.

PURIFICATION AND KINETIC PROPERTIES OF ATP: CITRATE OXALOACETATE LYASE FROM RAT BRAIN1

Abstract— A method for a partial purification of ATP:citrate oxaloacetate lyase from rat brain is described.The Lineweaver–Burk plots of velocity vs citrate concentration are biphasic in the presence of fixed concentrations of MgCl2. Therefore two values of Km, corresponding to low and high concentrations of citrate, can be determined. When MgCl2 is added in equimolar concentrations with citrate, a monophasic plot with one Km of 0.13 mm is obtained. The Km value for MgATP2‐ was independent of citrate concentration, being equal to 0.40–0.43 mm. The Km for CoA was 0.0007 mm.ADP and Pi are competitive inhibitors with respect to ATP. Ki for MgADP− is equal to 0.13 mm. dl‐isocitrate and cis‐aconitate are partially competitive inhibitors with respect to citrate with Ki values of 5.8 and 4.8 mm, respectively. α‐Ketoglutarate and pyruvate are noncompetitive inhibitors with respect to ATP and citrate, with Ki values equal to 9 and 45 mm, respectively.The physiological significance of these effectors for the regulation of citrate lyase activity in brain is discussed.

Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro.

1. Fatty n-acyl-CoA derivatives in the concentration range 5muM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 muM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.

A study of the kinetics of iron and copper binding to hen ovotransferrin

The kinetics of iron and copper binding to hen's-egg apo-ovotransferrin were studied by using citrate chelates of these metals at pH9.3 in borate buffer in the presence of bicarbonate. The kinetics of the absorbance change associated with the formation of the final product show a fast process, which is pseudo-first-order, where the reagents are in excess with respect to the protein, and the citrate concentration is higher than 25mM. At lower citrate concentration, the progress curves are clearly biphasic. There is marked dependence of the rate of the reaction on bicarbonate concentration, which may be interpreted as a displacement reaction of the ligand-metal-protein ternary complex. The kinetics have been interpreted in the framework of a reaction scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent colour development by displacement of the chelator by bicarbonate. The pH-dependence of this reaction supports the belief that tyrosine residues are involved in the process of iron-binding. The overall similarity of kinetics for iron and copper binding, notwithstanding their different co-ordination preferences, suggests that the process of metal-binding or chromophore development for the two metal complexes must be similar.

The regulation of chick ( Gallus domesticus) mucosal phosphofructokinase by ammonium and citrate ions

1. 1. A partially-purified preparation of phosphofructokinase from chicken jejunal mucosa was made and some of its properties determined. 2. 2. The enzyme had a pH optimum of about 7.8 and was strongly inhibited by ATP (1.5 mM). Fructose 6-phosphate saturated the enzyme at a concentration between 2.5 and 3.0 mM. 3. 3. Ammonium ions markedly stimulated enzyme activity, but the enzyme was less sensitive to citrate inhibition than expected, with lower concentrations of citrate (0.5 mM) stimulating activity. Some evidence for an interaction between the effects of ammonium and citrate ions was found. 4. 4. These results are discussed and compared with published findings for the mammalian enzyme.

Deoxyribonuclease activity in semen.

Summary. The methyl green assay method has been used to determine the activity of DNase I and DNase II in ram, bull, human, dog and rabbit seminal plasma. The activity of DNase II was higher except in the ram. The DNase I activity of ram seminal plasma was inhibited by low concentrations of citrate, zinc and cetyltrimethylammonium bromide ; this detergent also caused a reduction of DNase II activity. There was a stimulation of DNase I activity by 10 mm-citrate but not by concentrations less than 2 mm. Addition of antibiotics or shaking of ram seminal plasma with toluene did not affect the activity of either DNase. After deep freezing of ram semen there was a decrease of DNase I activity in the plasma and an increase of DNase I and II activity in extracts obtained from disintegrated spermatozoa. No significant change in DNase activity could be detected in the plasma of ram semen which had previously been subjected to cold shock.

A role for carnitine and long chain acylcarnitine in the regulation of lipogenesis

In the absence of citrate or other acetyl-CoA carboxylase activator, fatty acid synthesis from acetate or low concentrations of citrate in cell free preparations of rat liver proceeds at a very low rate, analogous to preparations obtained from starved or fat fed animals. Under these conditions, the addition of carnitine results in a further decrease in fatty acid synthesis. In contrast, when high concentrations of citrate are added, carnitine significantly stimulates lipogenesis from both acetate and citrate. The effects observed with carnitine at varying levels of citrate are related both to the availability of carnitine and CoA as well as the ratio of activities of carnitine acetyltransferase and acetyl-CoA carboxylase. In the absence of citrate, acetyl-CoA is shuttled into acetylcarnitine in the presence of carnitine, whereas at high concentrations of citrate and increased acetyl-CoA carboxylase activity, acetyl-CoA is preferentially utilized in the synthesis of fatty acids. In the latter case, carnitine enhances synthesis of long chain fatty acids perhaps by removal of a feed back inhibitor of acetyl-CoA carboxylase (palmitoyl-CoA) as well as providing an activator, palmitoylcarnitine. Both palmitoyl (+) carnitine and palmitoy (−) carnitine stimulate lipogenesis in high speed supernatant liver preparations. Palmitoyl-carnitine in addition stimulates acetyl-CoA carboxylase but does not effect the incorporation of malonyl-CoA into long chain fatty acids in high speed supernatant preparations, suggesting the site of action of long chain acylcarnitine at the acetyl-CoA carboxylase step. Palmitoyl-CoA inhibits a variety of key enzymes involved in the regulation of lipogenesis and glycolysis. Of those enzymes examined, glutamic dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase, malic enzyme, citrate lyase, citrate synthase, acetyl-CoA carboxylase, and carnitine acetyltransferase, only malic enzyme, malate and lactate dehydrogenases were not inhibited by palmitoyl-CoA. The remaining enzymes were all inhibited by low concentrations of long chain acyl-CoA and of those inhibited only citrate synthase, acetyl-CoA carboxylase, and carnitine acetyltransferase were relieved by palmitoylcarnitine. The results obtained suggest that perhaps the antagonistic effects of palmitoylcarnitine on inhibitions by palmitoyl-CoA may have some physiological significance and need not be necessarily categorized as non-specific.

The Interaction of Citrate With Aspirin and Benzoic Acid

Solubility studies were used to determine the nature and extent of the interaction between sodium citrate and aspirin. Because of the complex nature of this system, parallel studies of the solubilizing action of sodium citrate and acetate on benzoic acid were made for purposes of comparison. The acetate-benzoic acid reaction closely followed results expected from mass law considerations over a wide concentration range. However, the weak acid-citrate interactions could be described by the mass law only at low citrate concentrations, showing wide deviations at higher concentrations. It was apparent that the deviations were a function of many variables which included changes of solubility and Ka with the nature and concentration of the solution species. The fact that the interactions could not be rationalized by these changes alone at very high citrate concentrations suggested an association or associations among the dissolved species.