Acyl Phosphate Research Articles

Comparison of the HPLC-separated species patterns of phosphatidic acid, CDP-diacylglycerol and diacylglycerol synthesized de novo in rat liver microsomes (a new method)

The species pattern of phosphatidic acid was compared with that of CDP-diacylglycerol and diacylglycerol synthesized de novo by glycerol 3-phosphate acylation in a CoA ester-generating system in liver microsomes. The similarity of the species patterns of phosphatidic acid and CDP-diacylglycerol indicated that the CTP-phosphatidyl cytidylyltransferase showed no selectivity for individual species of its phosphatidic acid substrate. Since the species pattern of diacylglycerol deviated from that of phosphatidic acid, a slight acyl selectivity of the phosphatidic acid phosphohydrolase or a slight inhomogeneity of its substrate pool might be assumed. For the determination of the molecular species of CDP-diacylglycerol, a new method was developed. By incubation of CDP-diacylglycerol with oligonucleate 5'-nucleotidohydrolase (phosphodiesterase), phosphatidic acid was produced. The CDP-diacylglycerol-derived phosphatidic acid was methylated with diazomethane and then separated by reverse-phase HPLC in 15 molecular species.

Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis.

31P nuclear magnetic resonance spectroscopy of the phosphorylated tetrapeptide GlyGlyAspAla

AbstractThe model peptide glycylglycylaspartylalanine was phosphorylated in the NMR sample tube and characterized by 31P NMR spectroscopy. The pKa2 value of the acyl phosphate group in glycylglycyl(β‐aspartyl phosphate)alanine was determined to be 4.6 and the chemical shift was −6.45 ppm at low pH (1.5) and −1.27 ppm at high pH (8.0).

In Situ Measurement of the FT-IR Spectra of Phospholipid Monolayers at the Air/Water Interface

External reflection Fourier transform infrared spectroscopy has been applied to the in situ measurement of the infrared spectra of insoluble phospholipid monolayer films that were spread at the air/water interface. The spreading conditions for the phospholipids [1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)] were chosen to mimic those of liquid-condensed and liquid-expanded surface films, respectively. It is shown that the infrared reflection spectra can identify vibrations due to the hydrocarbon acyl chains, carbonyl ester, and phosphate groups for these monolayer films on water. Detailed examination of the frequencies and polarization properties of the C-H stretching region shows that the liquid-condensed DSPC acyl chains are in a rigid, mostly all-trans configuration, while the liquid-expanded DMPC acyl chains are highly fluid. The spectra demonstrate that the external reflection FT-IR experiment can differentiate between the physical conformation of monomolecular films when water is used as a reflective substrate.

53] Purification of the ATPase of Streptococcus faecalis

Publisher Summary This chapter focuses on the purification of the ATPase of Streptococcus faecalis . The vanadate-sensitive ATPase of Streptococcus faecalis is a novel bacterial ion pump, probably involved in K ÷ -transport. The gene for this ATPase has recently been cloned. The enzyme belongs to a new and growing class of prokaryotic ion-motive ATPases. Members of this class of enzymes exhibit a certain degree of sequence homology to eukaryotic ion-motive ATPases and undoubtedly bear an evolutionary relationship to them. This relationship also manifests itself in a number of properties these ATPases, whether eukaryotic or prokaryotic, have in common: (1) They form an acyl phosphate intermediate as part of the reaction cycle, (2) they are inhibited by micromolar concentrations of vanadate, and (3) they contain a major, and often single, polypeptide component of 60 to 120 kDa. The S. faecalis ATPase may serve as a useful model system for the study of such ion-motive ATPases as it can readily be prepared in homogeneous form in milligram quantities without the use of specialized equipment. ATPase activities are determined with a coupled enzyme assay that links the generation of ADP to the oxidation of NADH, which can be monitored spectrophotometrically at or near 340 nm. Measurements are best conducted with a dual-wavelength spectrophotometer, using the wavelength couple 550/366 nm.

13] Approaches to studying the mechanism of ATP synthesis in sarcoplasmic reticulum

This chapter focuses on the approaches to studying the mechanisms of ATP synthesis in sarcoplasmic reticulum. The synthesis of ATP is initiated by the phosphorylation of an aspartyl residue located in the catalytic site of the enzyme by Pi, forming an acyl phosphate residue. The part of the enzyme phosphorylated by Pi faces the outer surface of the vesicle. When the reversal of the Ca2+ pump was first described, it was thought that the energy derived from the Ca2+ gradient was used to drive this reaction. Regardless of the source of the isotope, [32P]Pi ([32p]orthophosphate) solutions usually contain radioactive contaminants, which represent a frequent source of error in measurements of phosphoenzyme formation from Pi. The chemical nature of the contaminants is not known. With purification, the level of contaminants decreases, but they reappear on standing. This observation suggests that radiolysis may promote the formation of odd radioactive phospho compounds. In the different experimental conditions tested, the rate of ATP hydrolysis was found to be always faster than the rate of ATP synthesis. Therefore, the data obtained with leaky vesicles and soluble Ca2+-ATPase indicate that the system must be able to conserve some of the energy released from ATP hydrolysis in a form that permits synthesis of ATP.

51] Genetics of Kdp, the K+-transport ATPase of Escherichia coli

This chapter describes the application of a variety of widely used techniques of bacterial genetics to the study of Kdp. Analogous genetic methods are already available for lower eukaryotes, such as yeast, but at present only a few are readily applied to higher eukaryotes. The rapid progress of genetics gives promise that the ease and extent of genetic dissection in bacteria and yeast will soon be possible in higher eukaryotes. Kdp is a complex of three-membrane proteins that form a K + -transport ATPase in Escherichia coli . Kdp belongs to the E l -E 2 class of transport ATPases, as shown by the formation of an acyl phosphate intermediate, and by homology to the Ca 2+ -ATPase and the Na + , K + -ATPase of animal cells, and to H + -ATPases of yeast and fungi. Genetic analysis has been important from the outset in the identification and characterization of the Kdp transport ATPase, in contrast to eukaryotic transport ATPases, where genetics has only recently added to the extensive information obtained by biochemical techniques. To analyze the kdp genotype of a strain and to construct strains with particular kdp genotypes for mapping, complementation analysis, or cloning, it is useful to be able to move kdp mutations from one strain to another. A convenient way to move them is cotransduction by bacteriophage P1 with a nearby marker. Three useful markers close to kdp are nagA, gltA, and nadA.

Further evidence for the existence of different diacylglycerol pools of the phosphatidylcholine synthesis in microsomes

Endogenous diacylglycerol and diacylglycerol, synthesized in vitro by glycerol 3-phosphate acylation, are not mixed and represent different substrate pools for the biosynthesis of phosphatidylcholine in microsomes of rat muscle, liver and lung. Freshly isolated lung microsomes contain 12–18 nmol diacylglycerol per mg protein, and incubation with CDPcholine showed a biphasic curve for the synthesis of phosphatidylcholine as lung microsomes enriched in diacylglycerol through the glycerol phosphate pathway. With respect to the synthesis of phosphatidylcholine, a part of this endogenous diacylglycerol (0.4–0.8 nmol/mg) was comparable with diacylglycerol de novo formed in vitro by glycerol 3-phosphate acylation. An increase in the relative proportion of de novo-fonned diacylglycerol in the total amount of diacylglycerol caused an increase in phosphatidylcholine synthesis by nearly the same factor. The apparent K m of the de novo-fonned diacylglycerol substrate for the choline phosphotransf erase was 10-times higher than the pool size of this diacylglycerol substrate in freshly isolated lung microsomes. The results supported the idea that the availability of this substrate type may be rate limiting for the de novo synthesis of phosphatidylcholine. As shown by use of the proteolytic technique measuring the mannose-6-phosphatase as lumenal control activity, the phosphatidylcholine synthesis from de novo-formed diacylglycerol and endogenous as well as exogenous diacylglycerol seems to be located on the cytoplasmic leaflet of the microsomal vesicles isolated from rat lung.

EVOLUTION OF HEPATIC CIRRHOSIS IN PEROXISOMAL DYSFUNCTION DISORDERS: THE SPECTRUM OF HISTOPATHOLOGIC ABNORMALITIES

Patients with peroxisomal dysfunction disorders lack peroxisomes or certain peroxisomal enzymes. Assay of activity of the peroxisomal membrane enzyme dihydroxyacetone phosphate acyl transferase (DHAP-AT) provides a biochemical test for identifying patients with peroxisomal disorders. We report 8 patients with a spectrum of peroxisomal disorders. All patients have dysmorphia, failure to thrive and hepatomegaly. Serum transaminase levels ore elevated and activity of DHAP-AT is reduced. All patients have other chemical abnormalities associated with peroxisomal dysfunction. One infant had Zellweger's cerebro-hepato-renal syndrome (CHRS) and died at age 10 months. The other patients presented with either a variant of Zellweger's CHRS or neonatal adrenoleukodystrophy (4), dicarboxylic aciduria (1), infantile Refsums' disease (1) or chrondrodysplasia punctata (1). Histopathologic changes in the liver include micronodular cirrhosis (2), fibrosis (4), and paucity of intrahepatic ducts (2). In addition, there was accumulation of hepatocyte hemosiderin in 1 patient and abundant deposition of glycogen in 3 patients. Peroxisomal disorders present with a variety of clinical problems. Infants with failure to thrive, hepatomegaly and suggestive dysmorphia should be screened by assaying DHAP-AT.

In vitro effects of chlorpromazine on glycerol 3-phosphate acyl transferase and 1-acylglycerol-3-phosphate acyltransferase in rat liver microsomes

The in vitro effect of chlorpromazine on rat liver glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferase was studied. Chlorpromazine decreased glycerol-3-phosphate acyltransferase activity in a concentration-dependent manner. The inhibition was competitive with respect to palmitoyl-CoA, while non-competitive with respect to sn-glycerol-3-phosphate. K i, was determined to be approximately 0.15mM with respect to both palmitoyl-CoA and sn-glycerol-3-phosphate. From these results, together with the inhibitory effect of amphiphilic anions and neutral detergents on the enzyme demonstrated by others, it was proposed that the hydrophobic moiety of chlorpromazine competes with acyl-CoA. The activity of 1-acylglycerol-3-phosphate acyltransferase was inhibited by excess of 1-acyl- sn-glycerol-3-phosphate. In the acceptor concentration range where the substrate inhibition was observed, chlorpromazine showed stimulatory effect on the enzyme activity. At lower concentrations of the acceptor, however, chlorpromazine produced marked inhibition of the enzyme activity. From the kinetic analysis, the inhibition was found to be uncompetitive with respect to acyl-CoA. It was found that the enzyme was more susceptible to the inhibitory action of chlorpromazine with unsaturated acyl-CoAs than with the saturated species, raising the possibility that chlorpromazine alters the molecular species composition of phosphatidic acid produced by the acylation reaction.

Glycerol 3-phosphate acylation in microsomes of type II cells isolated from adult rat lung

Glycerol 3-phosphate acylation was studied in type II cells isolated from adult rat lung. The process was found to be largely microsomal. In the microsomes phosphatidic acid is the main product of glycerol 3-phosphate acylation. Glycerol-3-phosphate acyltransferase is rate limiting in the phosphatidic acid formation by the microsomes. Type II cell microsomes incorporate palmitoyl and oleoyl residues into phosphatidic acid at an equal rate if palmitoyl-CoA and oleoyl-CoA are added separately. However, if palmitoyl-CoA and oleoyl-CoA are added as an equimolar mixture the unsaturated fatty acyl moiety is incorporated much faster. Under the latter conditions monoenoic species constitute the most abundant products of glycerol 3-phosphate acylation. The microsomes incorporate both palmitoyl and oleoyl residues readily into both the 1- and 2-position of phosphatidic acid, even when palmitoyl-CoA and oleoyl-CoA are added together. Assuming that both phosphatidic acid phosphatase and cholinephosphotransferase do not discriminate against substrates with an unsaturated acyl moiety at the 1-position and a saturated acyl moiety at the 2-position, the last two observations indicate that a considerable percentage of phosphatidylcholine molecules synthesized de novo may have a saturated fatty acid at the 2-position and an unsaturated fatty acid at the 1-position, and that remodeling at the 1-position may be important for the formation of surfactant dipalmitoylphosphatidylcholine. They also indicate that type II cell microsomes are capable of synthesizing the dipalmitoyl species of phosphatidic acid. However, since there is a preference for the acylation of glycerol 3-phosphate with unsaturated fatty acyl residues, the percentage of dipalmitoyl species in the synthesized phosphatidic acid, and thereby the percentage of dipalmitoyl species in the phosphatidylcholine synthesized de novo, will probably depend on the relative availability of the various acyl-CoA species.

The mechanism of nucleotide induced calcium translocation across sarcoplasmic reticulum membranes: Evidence for a non-translocated intermediate pool of calcium

Our previous data suggested that, in cardiac muscle sarcoplasmic reticulum fragments, GTP hydrolysis occurs by an alternative enzyme cycle of the Ca2+ ATPase which is insensitive to (Ca2+) and does not involve an acyl phosphate intermediate. Despite this, GTP induces the incorporation of calcium into a membrane pool that is not translocated to the vesicular lumen. The present study suggests that this GTP-induced intermediate calcium pool is identical to a modulable component of the calcium translocation process in that: it has an identical pH sensitivity; the initial incorporation of calcium in response to GTP eliminates the initial rapid burst and lag component of the typical ATP-induced calcium uptake curve when ATP is added during GTP-induced calcium accumulation. Instead, the addition of ATP during GTP-induced calcium accumulation results in the prompt onset of the linear phase of calcium translocation; GTP-induced calcium accumulation directly affects the pH sensitivity of subsequent ATP-induced calcium accumulation. We suggest that the intermediate calcium pool is in series with calcium translocation and is the site of the pH sensitivity observed in calcium flux.

Reaction of the anionic acetylation agent methyl acetyl phosphate with D-3-hydroxybutyrate dehydrogenase.

Methyl acetyl phosphate is a competitive inhibitor of the reduction of acetoacetate by D-3-hydroxybutyrate dehydrogenase. The material also irreversibly inactivates the enzyme. The kinetics of the inactivation are consistent with methyl acetyl phosphate acetylating the conjugate base of a hydrogen bond donor. Protection offered by a substrate analogue (methyl acetonylphosphonate) in the presence of coenzyme implicates reaction at the cationic active site. Reversible protection by the amino group reagent 2,3-dimethylmaleic anhydride suggests that methyl acetyl phosphate reacts with an amino group. Sulfhydryl reagents and acetyl phosphate, a poorer acetylating agent, do not inactivate the enzyme. The pH dependence of the inactivation suggests that the acetylation occurs at a site that has a pKa of 8.2. The utility of methyl acetyl phosphate and other acyl phosphate monoesters in reacting with lysines adjacent to cationic sites of enzymes, hemoglobin, and histones is noted.

ChemInform Abstract: Design and Synthesis of New Transition‐State Analogue Inhibitors of Aspartate Transcarbamylase.

AbstractDie dem gemäß Formelschema dargestellte Phosphonsäure (VII) und die ebenfalls synthetisierten Verbindungen (VIII) werden auf Inhibierung von Aspartat‐Transcarbamoylase untersucht.

Autophosphorylation of glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes.

Glyceraldehydephosphate dehydrogenase purified from rabbit skeletal muscle is auto-phosphorylated with MgATP. Half-maximal phosphorylation is achieved around 0.3 mM. The phosphorylation is Ca2+ independent. The phosphoenzyme complex is labile in alkaline conditions and stable in moderately acid media. The complex is readily hydrolyzed by 0.1 M neutral hydroxylamine, indicating the complex formed is a high-energy acyl phosphate. The phosphorylation is reduced by nicotinamide adenine dinucleotides, reduced form (NADH), glyceraldehyde 3-phosphate, and nicotinamide adenine dinucleotide (NAD+). The enzyme is also dephosphorylated by these metabolites although to a lesser extent by NAD+. Calsequestrin isolated from rabbit skeletal muscle inhibits the phosphorylation of the enzyme. The phosphoenzyme behaves as a kinase catalyzing the phosphorylation of proteins of Mr 80 000 and 72 000 found in the skeletal muscle terminal cisternae/triad preparation. This reaction is enhanced by NADH. The phosphate found in the protein substrate has been shown to be the same phosphate initially involved in the phosphorylation of glyceraldehydephosphate dehydrogenase.

Formation of an aspartyl phosphate intermediate in the reactions of nucleoside phosphotransferase from carrots

The nucleoside phosphotransferase from carrots forms N-phosphorylhydroxylamine when substrates are hydrolysed in the presence of hydroxylamine. Denaturation of the enzyme after short incubation with the substrates leads to a protein, in which, after reduction with [3H]NaCNBH3 and complete hydrolysis with 6 M HCl, labelled homoserine can be detected. The first experiment provides evidence for an activated phosphorylenzyme, the second experiment shows that the intermediate is an acyl phosphate formed by a nucleophilic attack of an aspartate beta-carboxylate group.

Ca 2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas

1. 1. Microsomes isolated from cat pancreas were incubated with [γ- 32P]ATP in the presence or absence of Ca 2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide absence of Ca 2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single microsomal protein with an apparent molecular mass of 77,000 dalton (77K) was found to be phosphorylated in a Ca 2+-dependent mechanism. 2. 2. Maximal phosphate incorporation into the 77K protein was observed at 10 −6mol/l[Ca 2+ and was 4-fold higher than in the absence of Ca 2+. 3. 3. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. 4. 4. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [γ- 32P]ATP and reached maximum at 9–10th min. 5. 5. The lack of effect of exogenous cyclic AMP, cyclic AMP-dependent protein kinase, calmodulin, the calmodulin antagonist trifluoperazine, leupeptin and the suppression of phosphorylation by some phospholipid-interacting drugs suggested that the 77K protein is a substrate for cyclic AMP- and calmodulin-independent, Ca 2+-activated phospholipid-sensitive kinase activity. 6. 6. Centrifugation of the pancreatic homogenate in a ficoll-sucrose densitu gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.

Nucleoside phosphotransferase from malt sprouts. II. Studies on the active site and the phospho-intermediate.

The phospho-intermediate formed in the reaction of the nucleoside phosphotransferase is an acyl phosphate, the phosphorus bound to the gamma-carboxylate group of a glutamic acid. Reduction of this intermediate with sodium cyanoborotritiide yields labeled 2-amino-5-hydroxyvaleric acid after hydrolysis of the protein. Nucleophilic trapping of the intermediate with hydroxylamine during the reaction with substrates leads to N-phosphohydroxylamine, which is the only reaction product at a higher concentration of hydroxylamine. Evidence is obtained from modification experiments that in addition to the carboxylate group a histidine is involved in the reaction. The pKa-value for the histidine derived from the photoinactivation of the enzyme is 7.6, indicating that this group forms a salt bridge to a carboxylate group, probably that group attacking the phosphorus. The acceptor nucleosides are bound only by hydrophobic interactions of the base, a conclusion obtained from fluorescence studies and quenching experiments. The hydrophobic interaction obviously does not involve pi-interactions to tyrosine and tryptophan residues, since their fluorescence is not affected by addition of nucleotide inhibitors. Modification of these residues leads only to unspecific inactivation. From the Scatchard plot of the titration of the enzyme with 1,N6-ethenoadenosine 5'-phosphoramidate, an efficient inhibitor (Kd = 1.2 X 10(-5) M), it can be concluded that there is only one binding site on the dimeric enzyme.

Nucleoside Phosphotransferase front Malt Sprouts. III. Studies on Metal Ion Requirement, Solvent Effects, Kinetics and Reaction Mechanism

The nucleoside phosphotransferase from malt sprouts contains one Mg2 per dimeric enzyme molecule. This cation can be removed by EDTA, while other chelating agents are less efficient. The metal-free apoenzyme is inactive. Activity can be restored to its initial value by Mg2 or Co2 and to a minor extent by Mn2, Zn2, Cu2, Ni2 and Fe2. Thermal stability is reduced when the metal is removed but can be restored by addition of Mg2. Adenosine 3',5'-phosphate (cAMP) and arsenate, strong competitive inhibitors, reduce the rate of inactivation by EDTA considerably but do not reduce the rate for the reconstitution with Mg2. We therefore conclude that the phosphate group interacts electrostatically with Mg2 and that the inhibitor is not bound to the apoenzyme. The Sp-isomer of adenosine 3',5'-thionophosphate is a hundred times stronger inhibitor than the Rp-isomer and ten times stronger than cAMP; this allowed us to determine the relative position of the Mg2 in the active site. The imidazolium cation, previously detected as an essential part of the active site, obviously forms a salt bridge to the carboxylate group which attacks the phosphorus opposite to the leaving alcohol group. This conclusion is derived from the fact that organic solvents increase the rate of formation of the phospho-intermediate considerably, while higher concentrations of salt decrease it strongly. In addition, the imidazolium cation seems to polarize the P = O bond and to stabilize the negative charge at the phosphoryl oxygen in the pentacoordinate transition state; this is followed from the pH-dependence of the hydrolysis reaction. The kinetic results reveal that Km does not represent a binding equilibrium, but is the ratio of the rates for decay and formation of the covalent intermediate, while kcat/Km is an indicator for the formation step of the acyl phosphate. On the basis of all these informations it should be allowed to formulate a reaction mechanism, in which all steps of the transferase reaction have to be microscopically reversible.

The effect of coronary artery occlusion and reperfusion on the activities of triglyceride lipase and glycerol 3-phosphate acyl transferase in the isolated perfused rat heart

Glycerol 3-phosphate acyltransferase (GPAT) and Triglyceride lipase (TGL) were measured in homogenates from non-ischaemic and ischaemic tissue from the isolated perfused rat heart. Ischaemia was produced by occlusion of the left descending coronary artery for 10 min. Compared to activities measured in tissue from normally perfused hearts, GPAT activity measured in tissue from the ischaemic area was considerably reduced. TGL activity in the ischaemic area was markedly increased compared to activity measured in normally perfused hearts. No change was seen in GPAT or TGL activity measured in tissue from the non-ischaemic area. The change in activities produced by ischaemia were prevented by pre-perfusion with the cardio-selective beta-antagonist Atenolol. Reperfusion of the ischaemic area resulted in TGL activity returning to the value measured in tissue from normally perfused hearts. However, GPAT activity, after 1 min of reperfusion, fell to a value lower than after 10 min ischaemia. The reperfusion-induced fall in GPAT activity was prevented by pre-perfusion with the alpha 1 antagonist Doxasozin. Pre-perfusion of the alpha 2 antagonist Yohimbine resulted in a prolongation of the increased TGL activity in the ischaemic area during reperfusion. All changes in enzyme activities were prevented by injection of 6 OH-dopamine 24 h before hearts were removed. These changes in enzyme activities show that during ischaemia there is an increased beta-adrenergic drive. On reperfusion the beta-adrenergic drive is removed but an alpha 1 adrenergic drive becomes apparent.