Giorgio Trombetta

Fructose-bisphosphate aldolase from rabbit muscle: A thermodynamic study on the formation on the enzyme-dihydroxyacetone phosphate complex

Abstract The influence of pH, ionic strength and temperature on the formation of the aldolase-dihydroxyacetone phosphate complex has been investigated. It has been shown that the formation of the complex is controlled by groups with a p K of 7.0 which may belong either to the substrate or to the enzyme. The pH dependence of the binding of the substrate is more complex above pH 7.0, and is not interpretable in terms of a few p K values. It has been confirmed that the increase of the ionic strength increases the dissociation of the complex. This shows that the interaction of the charged groups of the substrate and of the enzyme is a relevant feature in the formation of the complex. It has also been confirmed that the active site is positively charged in the entire pH range investigated. Both a decrease in the enthalpy and an increase in the entropy contributes to the formation of the complex; the entropy change, however, provides the major contribution.

The NAD domain and the predicted structure for aldolase: A word of caution

Abstract The proposal of E. Stellwagen [(1976) J. Mol Biol. , 106 , 903–911] that the structure of a protein can be predicted by sequence analysis provided that the protein specifically binds Cibacron blue F3GA, is not sound at least for muscle fructose bisphosphate aldolase. Contrary to the predictions we have shown that Cibacron blue does not interact directly with lysine 227 at the catalytic sites but with different sites which bind also ATP and fructose bisphosphate. We have shown also that aldolase binds 3.5 molecules of dye per subunit (dissociation constant 1.9 μ m ), too great a number to support the hypothesis that the binding of Cibacron blue is a specific indication of the presence of an NAD domain.

Divergent effects of filamin and tropomyosin on actin filaments bundling

Filamin increases and tropomyosin decreases the susceptibility of F-actin to form bundles of filaments in the presence of polyethylene glycol 6000. The two proteins, which are located in the leading edge and in the internal part of the cell, respectively, are thus likely to display divergent effects on the microfilaments into bundles transition in these two areas of the cell.

On the stiffness of the natural actin filament decorated with alexa fluor tropomyosin.

Natural, phalloidin-free, actin filaments were decorated with tropomyosin made fluorescent by reaction with alexa fluor (R) 488 C(5) maleimide. The elastic modulus by stretching of these filaments was then determined and found to span between 38.2 MPa and 61.48 MPa. We tried also to determine the yield strength of the same filaments in the laser light trap operated at 920 mW, the maximum power of the apparatus. Only two out of the 10 filaments tested were broken under these conditions, yield strength being 50.5 and 55 pN, respectively.

On the mechanics of the actin filament: the linear relationship between stiffness and yield strength allows estimation of the yield strength of thin filament in vivo

Comparison of the behaviour of actin filaments either modified with tetramethylrhodamine iodoacetamide or decorated with tetramethylrhodamine-phalloidin or with tropomyosin or with myosin subfragment 1 shows that, in all the cases, yield strength is linearly related to stiffness.

The stiffness of the crossbridge is a function of the intrinsic protein osmotic pressure generated by the crossbridge itself

A model is presented that makes it possible to determine the stiffness of the crossbridge from protein osmotic stress experiments. The model was elaborated while studying the osmotic properties of F-actin and of myosin subfragment-1 F-actin. These studies showed that the elastic modulus by bending of the monomer is directly related to the intrinsic protein osmotic pressure of the system. At a protein osmotic pressure of 1.8 x 10(5) dynes/cm2, the physiological protein osmotic pressure of frog skeletal muscle, it was found that the elastic moduli by bending of the monomer in F-actin and in the myosin subfragment-1 decorated F-actin are 6.5 X 10(7) and 3.3 X 10(8) dynes/cm2, respectively. The value of the elastic modulus by bending of the monomer in the myosin subfragment-1 decorated F-actin compares favorably with the values of the elastic modulus by stretching determined in skeletal muscle fibres.

The actin gelling activity of chicken gizzard α-actinin at physiological temperature is triggered by water sequestration

At 37°C, in the presence of 6% ( ) polyethylene glycol 6000, 30 nM α‐actinin from chicken gizzard induces the gelation of 12 μM actin. Static measurement shows that the addition of 30 nM α‐actinin increases the rigidity of the system from 23.5 to 54 . According to the theory of osmoelastic coupling, also large additives, such as the proteins of the cell sap, are able to cause an osmotic stress equivalent to that caused by polyethylene glycol. We thus conclude that, in vivo, α‐actinin acts as an actin gelling protein.

Fructose bisphosphate aldolase from rabbit muscle. A new, acid-labile intermediate of the aldolase reaction and the partition of the enzyme among the catalytic intermediates at equilibrium

Abstract By reaction of aldolase with dihydroxyacetone phosphate an acid-labile intermediate is formed, which is in rapid equilibrium with the eneamine intermediate. The equilibrium concentration of the eneamine + the acid-labile intermediates is constant between pH 5.5 and 7.5 and is not significantly different for native and for carboxypeptidase-treated aldolase. These data are in keeping with the view that the CH bond breaking and the CH bond forming at the C3 of dihydroxyacetone phosphate are affected to the same extent by the carboxypeptidase treatment. The formation of the acid-labile intermediate is reversed by the addition of hexitol bisphosphate or by the removal of the dihydroxyacetone phosphate present in the medium; both these reactions display a biphasic time course. The acid-labile intermediate disappears rapidly when the enzyme-substrate complex is oxidized by ferricyanide, in this case the biphasic behavior is not observed. This means that practically all the acid-labile intermediate is rapidly converted into the eneamine and becomes available for the condensation reaction. At the equilibrium the enzyme-fructose bisphosphate and the enzyme-triose phosphate complexes represent 74 and 26%, respectively, of the total complexes, the rate constants for the condensation and for the cleavage reactions being, respectively, 19.3 and 6.7 s −1 . These data support the view that the cleavage of the CC bond is the limiting step of the overall reaction.

Fructose 1,6-bisphosphate aldolase from liver: The absolute configuration of the intermediate carbinolamine

1. Introduction The classical studies of Rose et al. [ 1,2] on the stereochemistry of the aldolase reaction have shown that the enzyme controls the orientation of the alde- hyde approaching the active site as well as which face (face si) of the enolate ion is attacked. We complete here the description of the configura- tion of the enolate ion by proposing that the lysyl amino group of the active site [3 J attacks the ‘face si’ of the C-2 group of fructose bisphosphate with the intermediate formation of the (2R)-carbinolamine. 2. Materials and methods Fructose bisphosphate aldolase (D-fructose 1,6- bisphosphate D-glyceraldehyde 3-phosphate lyase, EC 4.1.2.13) from beef liver was prepared according to the procedure of Chappel et al. [4]. Only form II (spec. act. 0.8 I U/mg protein) was used in these experiments. Aldolase activity was measured spectro- photometrically [S] . Protein was measured assuming an absorbance of 0.89 at 280 nm for a 1 mg/ml solu- tion of liver aldolase [6]. [U-“C] Fructose bisphos- phate was purchased from the Radiochemical Centre, Amersham, England. N6-&Glycerollysine was prepared according to the procedure of Speck et al. [7]. N6 (2-Deoxy-2-glucitol) L-lysine (glucitollysine) and N6 (2-deoxy-2-mannitol) Llysine (mannitollysine) were prepared by condensa- tion of bromobutylhydantoin with either mannitol- amine or glucitolamine. The procedure was the same as for glycerollysine except that condensation was performed in methanol. 244 Mannitollysine, glucitollysine and glycerollysine were separated by chromatography on a Dowex 50-X8 (Na’-form) 200-400 mesh 1.1 X 80 cm column follow- ing the procedure of Moore and Stein [8]. The radioactive protein derivative was precipitated by the addition of 436 mg solid ammonium sulfate/ ml of solution. The precipitate was dissolved in 1 of M NaCI, dialysed against 0.15 M NaCl and against water. Radioactive determinations were made in a Packard Tri-Carl scintillation counter in 10 ml of Bray solution [9]. Protein hydrolysis was performed at 110°C for 48 h in evacuated sealed tubes. The hydrolysate was dried under reduced pressure in a rotary evaporator, dissolved and dried three times from water and finally dissolved in 2 ml of 0.1 M sodium citrate buffer, pH 3.42. 3. Results and discussion Liver aldolase (9 mg) was treated for 7 min at 2°C with 0.64 mM [U-14C]fructose bisphosphate (specific radioactivity 3 100 cpm/nmol) and 17 mM NaBH4 in the presence of 0.1 M sodium acetate buffer, pH 5.3. After treatment, catalytic activity was reduced to 30% and the radioactive substrate (192 000 cpm) was irreversibly bound to the enzyme. The protein was hydrolysed in 6 N HCl and the hydrolysate was submitted to chromatography on Dowex 50-X8 (Na’-form). Two radioactive peaks (fig.1) were eluted accounting for 12% and 68% respectively of the radioactivity (192 000 cpm) placed on the column. The first peak (23 000 cpm) was coincident with the authentic standard of glucitol- lysine and the second peak (130 000 cpm) was

On the stereospecific reduction of the aldolase-fructose 1,6 bisphosphate complex by NaBH4

Aldolase from rabbit muscle covalently binds fructose 1,6 bisphosphate after reduction with NaBH4. The reaction is stereospecific since after acid hydrolysis of the protein only N6(2-deoxy-2-glucitol) L-lysine is isolated. It is suggested that the lysyl amino group of the active site reacts with the face si of the C-2 group of fructose bisphosphate.