THE EFFECT OF METAL IONS, OXALATE AND AMINOACIDS ON THE ACTITIVITY OF PYRUVATE KINASE FROM CURIMBATÁ (Prochilodus lineatus) EPAXIAL MUSCLE

A research has been carried out on the effect of divalent ions, oxalate and aminoacids on the activity of pyruvatekinase (PK) purified from the epaxial muscle of the tropical fish curimbatá (Prochilodus lineatus). This preparation of PK is activated by Mgand Mn being sensitive to Cu, Co, Ni and Zn. Ni and Zn inhibit PK activity in about 70% and 90%, respectively. The activity of this PK preparation is inhibited in about 85% by 2mM oxalate. In regard to aminoacids, PK from curimbata’s epaxial muscle displays several characteristics of M2 isoenzymes, but is not inhibited by L-alanine.


Introduction
Piruvate kinase (E.C. 2.7.1.40.-Adenosine-5'-triphosphate: pyruvate phosphotransferase) controls the outflow of the glycolytic pathway and catalyzes the transfer of a phosphoryl group from PEP to ADP.Pyruvate is formed and ATP is generated concomitantly.
Allosteric properties of PK are important on the control of glycolysis regulation.They are dependent of cooperative effects, and of the action of allosteric inhibitors.All phosphotransferases nucleotide dependent need one divalent ion.Pyruvate kinase from several tissues need one monovalent and one divalent ion, almost always K + and Mg ++ (BOYER, 1962;MUNDAY et al., 1980).
Several authors (RANDALL and ANDERSON, 1975;OCAMPOS et al., 1987;ISANI et al., 1994) showed the inhibitory effect of divalent ions (Ca ++ , Co ++ , Cu ++ , Be ++ , Zn ++ on PK from fishes. Several authors show that the aminoacid effect on PK isozymes change with the species.Avian and mammalian PK of the L type are activated by Fru-1,6-P 2 and inhibited by MgATP or L-alanine.However, PK of the M2 type is less sensitive to activation by Fru-1,6-P 2 , but is inhibited by MgATP, L-alanine, and L-phenylalanine.M1 isoenzyme is less sensitive to regulatory influences (MUNDAY et al., 1980;INAMURA and TANAKA, 1982;ROBERTS and ANDERSON, 1985;ENGSTRON et al., 1987).OCAMPOS et al. (1987) showed that Lalanine activates PK from striated muscle of Mugil lisa and Chaetoditerus faber.L-alanine and L-phenylalanine inhibit PK of Anguila rostrata; Carassius carassius and Oncorhynchus kisutch (JOHNSTON, 1975;GUDERLEY and CARDENAS, 1981;ROBERTS and ANDERSON, 1985).ZAMORA (1989) verified that PK from icefish muscle was inhibited by MgATP and sodium oxalate and that this inhibition was partially reversed by MgATP.
In this report we wish to present results to improve the understanding of the kinetic properties of PK from epaxial muscle of curimbata (Prochilodus lineatus).

Materials.
Adenosine The muscle was obtained by dissection, washed in physiological solution at 4 o C, and frozen at -20 o C.
Purification.Muscle was minced and homogenized in 33 mM phosphate buffer (pH 6.5) containing 1 mM MgCl 2 , 0.1 mM DTT (1 g tissue: 9 ml of buffer).The homogenate was centrifuged at 12,000 x g for 30 min, at 4 o C. The pellet was discarded.Ammonium sulfate was added to the supernatant until reach 70% saturation.After centrifugation at 12,000 xg for 30 min (4 o C) the pellet was resuspended in the buffer described above.The resuspended enzyme solution was dialyzed on the same buffer for 8 h at 4 o C.
Ion exchange chromatography was done in one 1,4 x 20 cm phosphocellulose column equilibrated and washed on the above buffer.After the sample application the enzyme was eluted with a linear gradient of 0 to 0.5 M KCl in the same buffer, between tubes 29 and 32.
The flow rate was 4 ml/10 min.Fractions containing PK activity were precipitated with ammonium sulfate (70%) and used for kinetic studies.
Protein Estimation.Protein was determined as described by LOWRY et al. (1951) with bovine serum albumin as a standard.

Results and Discussion
PK from curimbata muscle is dependent of Mg ++ or Mn ++ .The plots of velocity x concentration of substrate resulted in hyperbolic curves for both Mg ++ or Mn ++ .
The highest stimulation seen with Mg ++ was observed with concentrations of 10 mM MgCl 2 (pH 6.5) and 5 mM (pH 7.5).The maximal rates in the presence of Mn +2 were seen with 1,0 mM (pH 6.5) and 2,5 mM (pH 7.5).Concentrations higher than 2.5 mM are inhibitory, this effect being stronger at pH 7.5.
Mn ++ is a substitute of Mg ++ , but the activation seen with Mn ++ is 33% at pH 6.5 and 60% at pH 7.5 of the activation seen in the presence of Mg ++ (FIGURE 1).ZAMORA (1989) observed 40% activation in ice-fish.PLAXTON and STOREY (1985) and BLAIR and WALKER (1984) observed 40-60% in molusc hepatopancreas and rat liver, respectively.Kinetic studies revealed that the cation forms one complex metal-nucleotide, which is the active form of the substrate.PK needs one divalent cation to perform its catalytic activity (BOYER, 1962), because the nucleotide substrate is the complex MgADP.Ca ++ is a strong inhibitor of PK from curimbata epaxial muscle in the presence of Mg ++ (FIGURE 2).The inhibition was of more or less 50% in the presence of 2,5 mM CaCl 2 .In the presence of Mn ++ , no inhibition was seen until concentrations of 5 mM were used.The same results were seen in Mytilus edulis (DE ZWAAN et al., 1975), Chaenocephalus aceratus (ZAMORA, 1989) and Dicentrarchus labrax (ISAN et al., 1994).
Generally Ca ++ competes with Mg ++ or Mn ++ .The lower inhibition seen with Mn ++ is due the fact that Mn ++ forms more stable complexes with ADP (ISANI et al., 1994).
In carp (Carassius carassius), L-phenylalanine effect was reverted by Fru-1,6-P 2 .This effect was not observed in salmon (Oncorhynchus kisutch).In these fishes this inhibition is not so strong.M2 isoenzyme of mammals is sensitive to inhibition by L-alanine and L-phenylalanine.This effect is partially reverted by Fru-1,6-P 2 .PK from curimbata muscle display many of the characteristics of M2 isoenzyme, but is not inhibited by L-alanine.
PK from fish and invertebrate tissues cannot be under the same classification as used for mammalian's PK.Each tissue has characteristic properties and biological functions which results in distinct isoenzymes and regulatory mechanisms.
Concentrations between 1 and 20 mM of MgATP result in inhibitions between 6 and 50% of PK from curimbata epaxial muscle.
Aminoacid and MgATP effects on PK activity are seen on FIGURE 4. Addition of 20 mM L-alanine in the assay mix did not result in any inhibitory effect on PK from curimbata.On several species of fishes, Lalanine has one inhibitory effect on muscle PK (SOMERO and HOCHACHKA, 1968;JOHNSTON, 1975), while in other species, L-alanine is one activator (OCAMPOS et al., 1987).