Effects of protein-modifying reagents on an isoenzyme of potato apyrase

The reconstituted glycolytic system described previously (Scopes, 1973) was used to simulate post-mortem glycolytic metabolism in muscle. The effects of the following factors have been investigated: ATPase (adenosine triphosphatase) amount, AMP deaminase amount, percentage of the phosphorylase in the a form and the effect of diluting the glycolytic enzyme complex as a whole. It was confirmed that the rate of metabolism was solely dependent on the amount of ATPase present and that various concentrations of the glycolytic enzymes had no effect over a wide range encompassing the variation found in anatomically different muscles. The extent of metabolism, represented by the value of the ;ultimate' pH, depended markedly on the amount of phosphorylase in the a form; as little as 1% of the a form resulted in a considerably lower pH than in its absence. To a lesser extent the amount of AMP deaminase also affected the ultimate pH, but this was probably only significant for comparisons of genetically distinct muscles with widely differing amounts of AMP deaminase. The reconstituted system behaved almost identically with regard to post-mortem glycolytic metabolism compared with intact muscle tissue. It is concluded that the controlling effectors found with the reconstituted system apply to intact muscle also.

Section: Discussionmentioning

confidence: 99%

Studies with a reconstituted muscle glycolytic system. The rate and extent of glycolysis in simulated post-mortem conditions

Scopes¹

1974

“…Platelet-rich plasma, free from erythrocytes, was prepared as described (Rittenhouse, 1983) in the presence of 0.5 M-PGE1 (Upjohn Company, Kalamazoo, MI, U.S.A.), and centrifuged at 3000g for 5min at room temperature. Platelets were suspended in plasma/Ca2+and P1free gel-filtration buffer (see below) (1 :4, v/v), pH 6.5 (Rittenhouse-Simmons & Deykin, 1976) to a concentration of 6 x 109/ml, and were incubated for 60min at 37°C in the presence of 3.3 units of purified potato apyrase/ml (Valenzuela et al, 1973), and either 3yiCi of [5,6,8,9,1 1,12,14,15-3H]-arachidonic acid/ml (72Ci/mmol) and 20-40iCi of carrier-free [32P]P,/ml (Rittenhouse, 1983), or with 1 uCi of 5-hydroxy[2-'4C]tryptamine binoxalate/ml to label the storage granule pool (Rittenhouse-Simmons & Deykin, 1976). Radioisotopes were purchased from New England Nuclear (Boston, MA, U.S.A.).…”

Section: Preparation Ofplateletsmentioning

confidence: 99%

Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP

Rittenhouse¹

1984

173

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.

“…An apyrase isoenzyme of high adenosine triphosphatase/adenosine diphosphatase ratio was prepared from the Pimpernel variety of potato obtained by clonal selection (Valenzuela et al, 1973). The enzyme had a specific activity of 1210pmol of Pi released/min per mg of protein for ATP, and of 100 for ADP (ratio 12).…”

Section: Apyrase Preparationmentioning

confidence: 99%

“…If ATP and ADP also bind to distinct amino acids at the active sites, as suggested by the different protective effect of these two substrates from inactivation by tetranitromethane (Valenzuela et al, 1973), it is conceivable that the interaction between substrate and enzyme-inhibitor complex may be also different.…”

Section: Ppi and T-butyl Diphosphate Have The Same K1mentioning

confidence: 99%

See 1 more Smart Citation

Hydrolysis of synthetic pyrophosphoric esters by an isoenzyme of apyrase from Solanum tuberosum

Campo¹,

Puente²,

Valenzuela³

et al. 1977

A highly purified isoenzyme of apyrase obtained from potatoes (Solanum tuberosum var. Pimpernel) exhibits a low specificity for the organic moiety of synthetic pyro- and triphosphates. Methyl di- and tri-phosphates were hydrolysed at higher rates than ADP and ATP, but their Km values were also higher. Steric hindrance at the carbon atom linked to the pyrophosphate chain decreases both binding and maximum rate, whereas length or polarity of the organic chain do not have systematic effects. t-Butyl diphosphate, inorganic pyrophosphate, adenosine 5′-[alpha, beta-methylene]triphosphate and adenosine 5′-[beta, gamma-methylene]triphosphate are competitive inhibitors of the hydrolysis of ATP and ADP.