ABSTRACT
Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16µM/min/mg, Km of 13mM, Kcat of 3.48µM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.
ABSTRACT
Prophenoloxidase (proPO) was purified from blood cells of the brown shrimp Penaeus californiensis by ultracentrifugation and dye affinity chromatography. The isolated proPO is a 114-kDa monomeric protein as determined by SDS-PAGE. This protein can be hydrolyzed by proteinases, producing a 107-kDa active phenoloxidase (PO). The isoelectric point for both protein forms was 7.35. The PO reaction using L-DOPA as substrate, has an optimum pH of 8, and was poorly inhibited by sodium azide, thiourea and EDTA, but strongly inhibited by diethyl thiocarbamate. According to the substrate affinity and inhibition characteristics, this phenoloxidase was classified as a tyrosinase-like phenoloxidase. Purified proPO was not activated by bacterial lipopolysaccharides or beta-glucans.
Subject(s)
Catechol Oxidase/blood , Enzyme Precursors/blood , Hemocytes/enzymology , Penaeidae/enzymology , Animals , Catechol Oxidase/antagonists & inhibitors , Catechol Oxidase/isolation & purification , Centrifugation , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors/antagonists & inhibitors , Enzyme Precursors/isolation & purification , Hydrogen-Ion Concentration , KineticsABSTRACT
Bacteria of the genera Vibrio, Pseudomonas and Aeromonas were isolated from the intestine of apparently healthy brown shrimp (Penaeus californiensis Holmes) cultured in a tidal pond. Species from these genera of bacteria have been reported as shrimp pathogens and have been involved in human gastrointestinal disorders related to seafood consumption. Isolation was done first in Marine broth, then in selective media (TCBS, Cetrimide and MacConkey). The oxidase negative strains were discarded as insignificant to shrimp culture. The identification of oxidase positive strains was based in morphological and colonial characteristics, biochemical capabilities, and both salinity and temperature tolerance. API 20E system and fatty acid analysis were also included. Three potentially pathogenic bacteria, Vibrio parahaemolyticus, Vibrio furnissii and Pseudomonas putida were isolated and identified from healthy shrimp intestine.
