A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

Inhibitors of microsomal oxidations in insect homogenates

1. Homogenates of insect tissues were assayed for enzymes capable of oxidizing p-nitrotoluene to p-nitrobenzoic acid. 2. Locust fat-body homogenate 10000g supernatant was an effective enzyme and required no added cofactors. 3. Homogenates of other insects or locust organs and 10000g sediment from locust fat-body were not active and inhibited microsomal oxidations carried out by locust fat-body or rabbit liver enzyme. 4. Inhibitory power was high in homogenates of whole flies and of fly heads or thoraces. 5. Inhibition appeared to involve both irreversible inactivation of enzyme and the removal of essential cofactors.     

Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.     

A comparison of the influence of potassium and ammonium ions on the phosphofructokinases from rabbit muscle and rat erythrocytes.

Phosphofructokinases from rat erythrocytes and rabbit muscle have been compared in their kinetic behavior with respect to monovalent cation activation and ATP inhibition. Both ammonium and potassium ions affect the muscle enzyme in a two-fold manner: they act both as activators and effectors. On the other hand only ammonium exerts the two-fold effects on the erythrocyte enzyme, while the potassium ions activate without affecting cooperativity. The lower ATP inhibition of muscle phosphofructokinase may be partially explained by the action of potassium ions on the cooperative behavior of the enzyme. The differences between the phosphofructokinases from erythrocytes and muscle in the potassium type-II activation and ATP inhibition represent an organ specifity. Furthermore, the inhibition constants for 2, 3-bisphosphoglycerate differ by 10-fold between the two enzymes.     

Some properties of phosphofructokinase from kidney cortex and their relation to glucose metabolism

1. Phosphofructokinase from rat kidney cortex has been partially purified by using a combination of isoelectric and ammonium sulphate precipitation. This preparation was free of enzymes which interfered with the measurement of either product of phosphofructokinase. 2. At concentrations greater than the optimum, ATP caused inhibition which was decreased by raising the fructose 6-phosphate concentration. This suggested that ATP reduced the affinity of phosphofructokinase for the other substrate. Citrate potentiated the ATP inhibition. 3. AMP and fructose 1,6-diphosphate relieved the inhibition by ATP or citrate by increasing the affinity of the enzyme for fructose 6-phosphate. 4. K(+) is shown to stimulate and Ca(2+) to inhibit phosphofructokinase. 5. The similarity between the complex properties of phosphofructokinase from kidney cortex and other tissues (e.g. cardiac and skeletal muscle, brain and liver) suggests that the enzyme in kidney cortex tissue is normally subject to metabolic control, similar to that in other tissues.     

Regulation of locust fat-body phosphorylase

1. Glycogen phosphorylase of locust fat-body was partially purified by differential centrifugation and dissociation from glycogen particles at two pH values. 2. Optimum activity was obtained at pH6.6-6.7. 3. The calculated apparent K(m) values for glycogen and glucose 1-phosphate were 0.08% and 10-13mm respectively. 4. 5'-AMP activated in the range 5mum-1mm. 5. Glucose 6-phosphate is a competitive inhibitor for the substrate glucose 1-phosphate (K(i)=1.7mm). 5'-AMP abolishes this inhibition. Glucose weakly inhibits (K(i)=25-30mm), but trehalose does not inhibit even at 100mm. 6. It is suggested that glucose 6-phosphate is a major regulator of glycogen phosphorylase activity in locust fat-body.     

Regulatory properties of phosphofructokinase 2 from Escherichia coli

Escherichia coli K12 contains two phosphofructokinases: phosphofructokinase 1, the most studied one, appears to behave as an allosteric enzyme, while phosphofructokinase 2 presents the features of a Michaelian enzyme. We show the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-phosphate. Although both phosphofructokinases of E. coli K12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo.     

A comparative study of the regulation of Ca2+ of the activities of the 2-oxoglutarate dehydrogenase complex and NAD+-isocitrate dehydrogenase from a variety of sources.

Ca2+ was shown to activate oxoglutarate dehydrogenase and NAD+-isocitrate dehydrogenase from heart and other rat tissues by markedly decreasing the Km values of the enzymes for their respective substrates [see Denton & McCormack (1980) FEBS Lett. 119, 1-8]. Similar effects of Ca2+ were observed in the present study with both enzymes from other vertebrate sources (pigeon, trout, frog and human heart), but not with the enzymes from blowfly or locust flight muscle, or potato or Escherichia coli. In contrast, the Km values of the oxoglutarate dehydrogenases were affected by ADP, ATP and H+ to a similar extent in every case, except for the enzyme from E. coli, which was not sensitive to regulation by these agents.     

Coproporphyrinogenase in tobacco (Nicotiana tabacum L.)

1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.     

Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis

1. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for phosphofructokinase. The properties of this enzyme were found to be similar to those of the same enzyme from other tissues (e.g. cardiac muscle, skeletal muscle and brain) that were previously investigated by other workers. 2. Low concentrations of ATP inhibited phosphofructokinase activity by decreasing the affinity of the enzyme for the other substrate, fructose 6-phosphate. Citrate, and other intermediates of the tricarboxylic acid cycle, also inhibited the activity of phosphofructokinase. 3. This inhibition was relieved by either AMP or fructose 1,6-diphosphate; however, higher concentrations of ATP decreased and finally removed the effect of these activators. 4. Ammonium sulphate protected the enzyme from inactivation, and increased the activity by relieving the inhibition due to ATP. The latter effect was similar to that of AMP. 5. Phosphofructokinase was found in the same cellular compartment as fructose 1,6-diphosphatase, namely the soluble cytoplasm. 6. The properties of phosphofructokinase and fructose 1,6-diphosphatase are compared and a theory is proposed that affords dual control of both enzymes in the liver. The relation of this to the control of glycolysis and gluconeogenesis is discussed.     

The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofructokinases

The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-P2 show identical effects on the allosteric kinetic properties of both enzymes. Fructose-2,6-P2 counteracts inhibition by ATP and citrate and decreases the Km for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The Km values of liver and muscle phosphofructokinase for fructose-2,6-P2 in the presence of 1.25 mM ATP are 12 milliunits/ml (or 24 nM) and 5 milliunits/ml (or 10 nM), respectively. At near physiological concentrations of ATP (3 mM) and fructose-6-P (0.2 mM), however, the Km values for fructose-2,6-P2 are increased to 12 microM and 0.8 microM for liver and muscle enzymes, respectively. Thus, fructose-2,6-P2 is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-P2. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-P2, but the rates become linear in the presence of higher concentrations of fructose-2,6-P2. Fructose-2,6-P2 also protects phosphofructokinase against inactivation by heat. Fructose-2,6-P2, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.     

Arginine metabolism in insects. Role of arginase in proline formation during silkmoth development

1. Ornithine delta-transaminase (l-ornithine-2-oxo acid aminotransferase, EC 2.6.1.13) and Delta(1)-pyrroline-5-carboxylate reductase [l-proline-NAD(P) 5-oxidoreductase, EC 1.5.1.2] were demonstrated in fat-body and flight-muscle tissues of the silkmoth Hyalophora gloveri. Arginase (l-arginine ureohydrolase, EC 3.5.3.1) is also present in these tissues. 2. Arginase, ornithine transaminase and pyrroline-carboxylate reductase are generally considered to make up the catabolic pathway for the conversion of arginine into proline. The conversion of l-[U-(14)C]arginine into [(14)C]proline by intact fat-body tissue was used to show that the enzymes in insect fat body also function in this capacity. 3. Of the three enzymes of the catabolic pathway, only arginase increased during adult development and the increase coincided with the emergence of the winged adult moth. Since proline appears to be a major substrate utilized in insect flight metabolism, the increase in arginase activity at this stage suggests a major role for arginase in proline formation.     

Plasma membrane adenosine triphosphatase of oat roots: activation and inhibition by mg and ATP

ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L. cv. Goodfield) roots was examined in the presence of various concentrations of MgCl(2) and ATP. A Mg(2+): ATP ratio of about 1 was required for maximal activity regardless of the concentrations used; the optimum concentration for both Mg(2+) and ATP was 9 mm. Based on the ATPase activity at different concentrations of complexed Mg.ATP and free ATP, it is concluded that Mg.ATP is the true substrate of this enzyme.Under certain experimental conditions, high concentrations of MgCl(2) and ATP inhibited the plasma membrane ATPase. On the basis of the relative amounts of free and complexed ATP and Mg(2+), it was found that the different moieties caused different amounts of inhibition. Free ATP inhibited the ATPase at concentrations in excess of 2 mm. Mg.ATP concentrations above 11 mm inhibited the enzyme. Free Mg(2+) caused only a slight inhibition of the ATPase.The Km for Mg.ATP was found to vary from 0.64 to 1.24 mm depending on the experimental conditions. This variation is thought to be due to variable amounts of Mg.ATP, which serves as an inhibitor as well as the substrate, and free ATP, which also inhibits the enzyme.     

Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5-10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45-60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP(3) (F-IP(3)) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP(3), activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1h is sufficient to increase the lipid concentration.     

Pig kidney phosphofructokinase. Purification to homogeneity and qualitative basic characterization.

Phosphofructokinase has been purified from pig kidney by extraction with phosphate buffer at pH 8, followed by alcohol treatment, affinity chromatography on matrix-bound Cibacron blue F3G-A, and gel chromatography on Sepharose 6B. Using sodium dodecyl sulphate electrophoresis the enzyme was found to be homogeneous and to have a specific activity of about 80 units/mg protein. Like other phosphofructokinases, at pH 7.0 the enzyme exhibits a sigmoidal dependence in its activity on the fructose 6-phosphate concentration and is strongly inhibited by ATP. The degree of citrate inhibition is influenced by the concentration of the two substrates. ATP strengthens and fructose 6-phosphate relieves the inhibition by citrate. AMP and cAMP are able to overcome the ATP inhibition. The ADP activation curve is biphasic. The molecular weight of the subunit of pig kidney phosphofructokinase was determined to be 88 000 by means of sodium dodecyl sulphate electrophoresis.     

The effects of vanadate and molybdate on cathepsin D; relationship to ATP activation of lysosomal proteolysis

The effects of the phosphate analogues, vanadate and molybdate, on the ATP-activated enzyme, cathepsin D, were investigated. Both were found to inhibit proteolysis but this appeared to be the result of non-specific interactions with the protein substrates which result in precipitation, rather than interactions with the enzyme. Inhibition of proteolysis was induced by the same concentration of inhibitors as that which induced precipitation (measured by turbidity), and was dependent on the concentration of substrate. Precipitation did not occur at neutral pH but was maximal below pH 5. High concentrations of salt (greater than 1M KC1) prevented precipitation of proteins by vanadate and molybdate and under these conditions little inhibition of proteolysis was observed even at high inhibitor concentrations. Nonetheless, ATP was found to activate proteolysis catalyzed directly by lysosomal enzymes at acid pH, while vanadate and molybdate inhibited proteolysis in this system and induced precipitation of substrate. These results indicate that inhibition of proteolysis at acid pH by vanadate (or molybdate) has no relationship to inhibition of proteases and/or ATP dependence of such enzymes. However, direct activation of cathepsin D in lysosomes by ATP remains a viable hypothesis.     

Modulation of muscle phosphofructokinase at physiological concentration of enzyme

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.     

Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.     

The effect of the interdependence of protein and bile salt concentrations on the measurement of cerebroside sulphate sulphatase activity in human leucocyte and fibroblast extracts

The previously observed differences in properties of human leucocyte and fibroblast cerebroside sulphate sulphatase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) measured in vitro have been found to be due to subtle differences in incubation conditions. Maximum enzyme activity was observed with either crude sodium taurocholate or with pure sodium taurodeoxycholate. The optimum bile salt concentration of the enzyme in leucocyte or fibroblast extracts, but not the pure ox liver enzyme, was critically dependent on protein concentration. At low concentrations of the latter (less than 0.1 mg/ml), maximum activity was observed at taurocholate concentrations less than 0.5 mg/ml; at protein concentrations greater than 0.20 mg/ml substantially more bile acid (more than 1.3 mg/ml) was required to stimulate maximum activity. Addition of Triton X-100 or bovine serum albumin to the incubation mixtures increased the optimum taurocholate concentration. The dependence of the bile salt optimum on protein concentration appears to be related to the binding of the lipid substrate to membranous protein present in the tissue extracts. Release of the bound lipid is effected either by increasing the bile salt concentration or by adding Triton X-100. In the presence of excess bile salt human leucocyte, fibroblast and liver cerebroside sulphate sulphatase activity is stimulated by Triton at low protein concentrations; under identical conditions the pure or crude ox-liver enzyme is substatially inhibited. Our data also show that cerebroside sulphate sulphatase activity measured in extracts from leucocytes and fibroblasts, the tissues normally used to effect a diagnosis of metachromatic leucodystrophy, is the result of a complex interaction of bile salt, protein, Triton X-100 and probably the substrate itself. Any slight alteration in any of those factors, without a corresponding change in any or all of the others, can have a marked effect on the measured enzyme activity, and may lead to errors in the diagnosis of metachromatic leucodystrophy.     

Comparative studies on muscle AMP-deaminase--II. Regulation by monovalent cations, ATP and orthophosphate of the enzyme from hen, frog and pikeperch muscle.

1. Michaelis constants, maximum velocity and pH-dependence of the reaction catalysed by homogeneous AMP-deaminase preparations from hen, frog and pikeperch skeletal muscle were compared, as well as the influence of monovalent cations, ATP and inorganic phosphate. 2. ATP was found to activate the enzymes in the absence of K+ and at optimum (150 mM) KCl concentration. 3. Absolute dependence on potassium ions and considerable dependence of Km and Vmax on the kind of monovalent cation present in the medium were found for pikeperch enzyme.