Salivary apyrase of Rhodnius prolixus. Kinetics and purification

Abstract

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.

An Insight into the Sialotranscriptome of Triatoma matogrossensis, a Kissing Bug Associated with Fogo Selvagem in South America

“…59 Apyrases are diphosphohydrolases responsible for hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and P i , inhibiting platelet function by decreasing ADPinduced platelet aggregation, thus facilitating acquisition of the blood meal by hematophagous insects. 60 In triatomines, biochemical characterization of salivary apyrase activity was described for R. prolixus 61 and T. infestans.…”

Exploring the sialome of the blood-sucking bug Rhodnius prolixus

“…ADP at the submicromolar concentrations normally found in the plasma decreases the threshold for collagen-induced platelet aggregation. At these ADP levels, salivary apyrase is not an efficient scavenger of ADP due to its K m at >20 lM (Sarkis et al, 1986), but the salivary RPAI, with a nanomolar affinity for ADP, efficiently scavenges the nucleotide and raises the threshold concentration for collagen-induced aggregation . Pallidipin produces a similar inhibition of platelets and may act by the same mechanism (Noeske-Jungblut et al, 1994;Haendler et al, 1996) despite claims of its activity on the collagen receptor.…”

“…When probing in their victim's skin for blood, Rhodnius inject a salivary cocktail that contains apyrase, an enzyme that hydrolyses ADP, a nucleotide released by injured cells and a potent inducer of platelet aggregation (Sarkis et al, 1986). Additionally, a lipocalin with very high affinity for ADP mops up any residual nucleotide (Francischetti et al, 2000).…”

Patrones electroforéticos de hemoproteínas salivares (nitroforinas) de Rhodnius colombiensis y Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae)

“…En los triatominos se han encontrado varias sustancias relacionadas con estas funciones. En el caso del género Rhodnius, hasta el momento se han reportado ocho proteínas salivares: tres factores que contrarrestan la agregación plaquetaria, llamados inhibidores de agregación de R. prolixus (RPAI 1-3) (proteínas lipocalinas) (4,5); una apirasa que se encarga de hidrolizar el ADP bloqueando la agregación plaquetaria (4,6), y, por último, las nitroforinas (NPs), un grupo de cuatro hemoproteínas denominadas NP1 a NP4 según su relativa abundancia en las glándulas salivares (7). Las nitroforinas se pueden agrupar en dos conjuntos con base en las relaciones de sus secuencias de aminoácidos; es así como NP1 y NP4 son idénticas en un 90%, y NP2 y NP3 son idénticas en un 80% (4,7,8).…”