Allosteric regulation of 6-phosphofructo-1-kinase activity of fat body and flight muscle from the bloodsucking bug Rhodnius prolixus

Abstract

ABSTRACT6-phosphofructo-1-kinase (phosphofructokinase; PFK) activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues -flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 ± 1.4 U/mg) than in fat body extracts (3.6 ± 0.4 U/mg), although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5) and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.

Lactate favours the dissociation of skeletal muscle 6-phosphofructo-1-kinase tetramers down-regulating the enzyme and muscle glycolysis

“…This fact augments the relevance of the effects observed on PFK, since the formation of lactate occurs simultaneously with the acidification of the intracellular milieu [1][2][3][4][5][6][7], which turns lactate into a potent inhibitor of PFK and possibly of the glycolytic flux. PFK loses most of its regulatory properties at pH 8.2, when the tetrameric conformation of the enzyme is favoured and the enzyme works at its maximal velocity [10][11][12][13][14][15][16][17]33]. Our results show that at pH 8.2 PFK is unaffected by lactate, suggesting that the effects of the ligand occur through modulation of the enzyme's allosteric regulatory properties.…”

“…The parameters for ATP were calculated fitting eqn (3) to the experimental data presented in Figure 2 of PFK dissociate into less active dimers [10,13,14,[33][34][35]. In addition, it is well described that PFK is more susceptible to dissociation in acidic medium (a condition that potentiates the effects of lactate) being stabilized in the tetrameric conformation at pH 8.2 [15,36], a condition that abolishes lactate-induced inhibition of the enzyme.…”

“…The present study does not resolve this problem, but makes it clear that lactate is a PFK modulator. The inhibition promoted by lactate of PFK activity is counteracted by several physiological signals such as an increase in F2,6BP, the action of PKA and the association of the enzyme with f-actin, which is mediated by many other signals [17,[19][20][21][22][33][34][35][36][40][41][42][43][44][45][46]. Therefore lactate may act as an allosteric modulator of PFK, whose final effect depends on several other regulators of the enzyme.…”

Unique PFK regulatory property from some mosquito vectors of disease, and from Drosophila melanogaster

“…A scan of the activity was performed in the pH range of 6.0 to 9.0 in order to determine the optimum pH, which was in the range 8.0-8.5. Nevertheless pH 7.4 was used as this was the pH that was previously described to be the one where the enzyme is most sensitive to allosteric regulation [ 8 ].…”

“…Citrate does not inhibit PFK in any of these organisms. Other investigations have confirmed the absence of citrate inhibition in insects [ 7 , 8 ], but no other distinguishing regulatory features of insect PFK has been demonstrated. Although dipterans are of major importance in epidemiology, no research on the regulatory properties of PFK from any flight vector has been published to date.…”