Caffeine inhibition of glycogen phosphorylase from Mytilus galloprovincialis mantle tissue.

A different caffeine inhibition of both phosphorylated and unphosphorylated forms of glycogen phosphorylase from Mytilus mantle has been demonstrated. Caffeine increases the allosteric constant of phosphorylase b 30-fold, acting as an allosteric inhibitor (nH = 2) of mixed type with respect to inorganic phosphate (Pi) and AMP, and of single competitive type with respect to glycogen. The Mytilus phosphorylated form is also caffeine inhibited through competitive inhibition in relation to Pi and glycogen. In this case, the inhibitor does not modify the allosteric constant (near 2), neither does it display allosteric effects (nH = 1). The results demonstrate the notable modification of the nucleotide site promoted by the phosphorylation process and the existence of a functional inhibitory nucleoside site in Mytilus phosphorylase.

Modification of kinetic parameters of glycogen phosphorylase from mantle tissue of Mytilus galloprovincialis by a phosphorylation mechanism.

Initial rate and affinity studies on mantle Mytilus phosphorylase a were carried out in order to find possible differences in its kinetic properties with respect to phosphorylase b. Phosphorylase a was not stimulated for any AMP concentrations. Michaelis constants (Km) are 0.05 mg/ml glycogen, 1.15 mM inorganic phosphate and 1.50 mM glucose-1-phosphate. The Kms for the substrates, in the direction of glycogen breakdown, are enhanced by non-saturating concentrations of cosubstrate, without reducing the apparent maximum velocity. First order and hyperbolic kinetics and values of the allosteric constant smaller than 2 were observed. These results suggest a catalytic mechanism different to that shown for mantle Mytilus phosphorylase b.

Development of a method for the analysis of nucleotides from the mantle tissue of the mussel Mytilus galloprovincialis.

Reversed-phase HPLC was applied to obtain a sensitive and efficient means for quantitating nucleotides in the mussel Mytilus galloprovincialis. We obtained a good separation of adenylic, guanylic, uridylic and cytidylic nucleotides. Adenine nucleotides play a critical role in the regulation and integration of cellular metabolism; particularly in the mantle tissue in the mussel, they are involved in the regulation of the enzyme glycogen phosphorylase, a key enzyme in the transfer of bioenergetic reserves (glycogen) to gametogenic development; it is of great importance to have a measure of the concentrations in vivo during the reproductive cycle of the organism. Different elution conditions were tested: isocratic versus step gradient elution, different mobile phase pH and the type and proportion of ion-pairing agent added to the mobile phase. The best method was selected and the separation and accurate determination of adenine, citidine, guanine and uridine nucleotides was accomplished within a 20-min run, with UV-Vis detection (254 nm).

Amylase on Pecten maximus (Mollusca, bivalves): protein and cDNA characterization; quantification of the expression in the digestive gland.

The digestive enzyme alpha-amylase in Pecten maximus has been purified from the digestive gland, where it is present as two isoforms, In order to gain information on its structure and regulation, a digestive gland cDNA library, constructed in lambda phage Zap II (Stratagene, La Jolla, Calif., U.S.A.), was screened with a shrimp alpha-amylase cDNA probe. Only 0.02% of the clones were positive, and the longest clone, having a size of 1700 bp and identical to that of the mRNA, was fully sequenced. It contains the complete cDNA coding frame for one of the amylase isoforms of P. maximus. The deduced protein sequence is 508 amino acids long, with a putative 18 amino acid, highly hydrophobic signal peptide and a mature enzyme of 489 residues. The molecular weight corresponds to 54,500 Da and the calculated isoelectric point is 6.76. Locations of conserved sequences confirms the high level of similarity with the other members of the family.