PFK activity from fat body and flight muscle homogenates were measured as previously described Sola-Penna et al.
Polyclonal antibody raised against mammalian PFK was obtained in our laboratory Meira et al. IgG was colleted from 30 mL blood extracted at day 45, after centrifugation by 30 minutes at rpm.
The PFK activity from R. Figure 1A shows the activity of the enzyme measured in different pH, at fixed ATP and fructosephosphate concentrations 2 and 5 mM, respectively. The activity on both extracts, after normalized by the maximal activities obtained Fig. At pH 7. In order to compare the 6-phosphofructokinase activity from R.
The kinetic parameters were determined measuring the initial velocity of enzyme catalysis in the function of substrate concentration. All velocity measurements were performed following the product formation in the function of time. Figure 2 shows the curves of the initial velocity of PFK versus fructosephosphate concentration for the both tissues.
The enzyme velocity responds in an allosteric manner to fructosephosphate and the parameters of the equation 1 were best fitted to the experimental data. In addition, we observed that the K for fructosephosphate is lower in the flight muscle comparing to the fat body 0. Figure 3A shows that the enzyme responds in avery similar pattern to lower concentrations up to 3 mM of its other substrate, ATP.
In both extracts, the enzyme initial velocity responds in an allosteric manner to ATP. The parameters shown in Table I , obtained with the best fitting of experimental data to equation 1, revealed that the muscle homogenate, once again with a higher specific PFK activity, also has the lower K 0.
At higher concentrations Fig. However, the best fitting of experimental data to equation 2 revealed a lower K i for ATP at higher concentrations to the muscle enzyme, as shown in Table I. Although both extracts presented some degree of cooperativity toward ATP, we were unable to identify significant differences between them.
In different tissues, PFK is submitted to a tight regulation, as a result of interactions between substrates and several allosteric inhibitors and activators. Citrate is described as potentiating the inhibitory effects of ATP in several animal tissues, providing an explanation to the glucose-fatty acid-ketone body cycle. However, other allosteric effectors, such as nucleotides, were able to markedly affect PFK activity. Figure 5 shows that ADP is able to increase the enzyme activity on both extracts, but at different potencies.
At 5 mM ADP, fat body PFK activity was 6 times higher than that observed in the absence of effector, compared to an activation of 3 times found in the muscle. Differences between fat body and flight muscle became evident again when the allosteric regulator studied was AMP Fig. This effector was able to activate PFK on both extracts, but the pattern found was dependent on the conditions tested.
However, the same experiment performed with the flight muscle homogenates showed that AMP was able to activated PFK, but no differences were observed when the experiment was performed at different ATP concentrations Fig.
This dependency on ATP concentration was also found on the effects of fructose-2,6-bisphosphate, one of the most potent activators of PFK already described. At the saturating stimulatory ATP concentration 2 mM , fructose-2,6-bisphosphate was unable to activate the muscle homogenate activity Fig. However, at the inhibitory ATP concentration 5 mM , fructose-2,6-bisphosphate promotes a very potent activation of the enzyme in both tissues Fig.
For better visualization of the fructose-2,6-bisphosphate activation on PFK activity of the insect tissues, the data presented on panels A of figures 7 and 8 were recalculated relative activation Fig. As it can be seen, this activation is more pronounced on fat body 10 times than in flight muscle 4 times. As it is shown in Figure 9 , probing insect tissues extracts with antibodies against muscle PFK from rabbit caused the recognition of a specific band after electrophoresis.
Although both lanes presented staining, it was markedly higher in fat body homogenates, in all western-blotting assays performed. By comparison, and the electrophoretic mobility with several standard proteins and rabbit muscle PFK, we were able to calculate to these bands a molecular mass of 86 kDa per monomer data not shown. It is reasonable to consider carbohydrate oxidation as an important metabolic process in the flight muscle of insects, especially those used to short-range flight activity, and that a tight glycolytic control of PFK activity would then be required on this tissue.
It has been already shown in one such insect, Rhodnius prolixus , a significant decrease on muscle glycogen storage during flight Ward et al. According to our data, PFK activity in the flight muscle of R. ATP presents inhibitory effects at concentrations above 3 mM, higher than those described as inhibitory for other insects Walker and Bailey , Holden and Storey , but with an estimated Ki within the near-physiological range described previously Wegener et al.
It is possible that, in muscle, PFK activity remains mostly inhibited, and dependant on the activation promoted by other effectors. Curiously, in our experiments fructose-2,6-bisphosphate showed little or no effects at lower ATP concentrations, suggesting that the mechanism of activation by the compound is counteracting the inhibitory effects of ATP. A similar pattern was found on the fat body homogenates, although with some singular characteristics. This tissue presented higher calculated K 0.
While the highest levels of relative PFK activity were always obtained with muscle homogenates, the total extent of activation promoted by ADP, AMP and fructose-2,6-bisphosphate was markedly higher on fat body.
This sensitivity to allosteric effectors suggests a tightly regulated glycolytic pathway, which may be consistent with physiologic roles of the fat body. KineMage currently not supported The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pink tint and Subunit 2 in pink.
Two side chains in each subunit are shown, those of Glu red and Arg cyan , which are part of the F6P binding site in the T and R states, srespectively see below. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu and Arg in both subunits are red and cyan as before only the Ca and Cb atoms of the Arg side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed. The T state enzyme binds the inhibitor 2-phosphoglycolate gold; "PGC" , a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate PEP.
Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues 2 arg and 1 Lys; not shown.
In the high-activity R state, the positively charged side chain of Arg forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P white dashed lines , an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect. F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P gray; viewed by clicking on "F6P site". What happens to the central polypeptide helical segment residues in the R to T transition?
What does this do to the relative positions of the negatively charged Glu and the positively charged Arg ? Click on "F6P site". What influence would the absence of the positive charge of Arg have on the binding of F6P?
Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center.
Slide the "zoom" slider to enlarge the view. At one time, the negative charge of Glu was thought to have a negative effect on F6P binding in the T state.
This idea has not been supported by site-directed mutagenesis experiments [14]. The RA mutation caused a fold decrease in F6P binding.
Inherited erythrocytes PFK deficiency is associated with myopathy and hemolysis Tarui disease [15]. Phosphofructokinase 3D structures. Correct answer: Phosphofructokinase-1 PFK Explanation : While phosphofructokinase-2 is responsible for creating fructose-2,6-bisphosphate, this molecule will actually upregulate the enzymatic activity of phosphofructokinase-1 PFK Example Question 5 : Glycolysis Regulation.
Glycolysis is an energy producing process that breaks down glucose. In glycolysis, feedback regulation is seen in which of the following examples?
Possible Answers: Fructose-1,6-bisphosphate inhibiting pyruvate kinase. Explanation : Feedback inhibition is a process by which the products of a reaction or series of reactions slows, stops or inhibits one of the previous reactions in the process, thereby controlling the rate of reaction, and rate of formation of the products. Copyright Notice. View Biochemistry Tutors. Alexis Certified Tutor. Nathaniel Certified Tutor. Josephine Certified Tutor. Report an issue with this question If you've found an issue with this question, please let us know.
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