Enrico Grazi

[26] 6-Phosphogluconate dehydrogenase—Crystalline

Publisher Summary This chapter discusses the determination of crystalline 6-phosphogluconate dehydrogenase. The method of determination of enzymatic activity is based on the spectrophotometric measurement of reduced triphosphopyridine nucleotide (TPNH) + H + formed during oxidation and decarboxylation of D-gluconate 6-phosphate to D-ribulose 5-phosphate and CO 2 . In the course of purification procedure, the dried yeast preparation (50 g) is suspended in 150 ml of 0.1 N NaHCO 3 . After 5 hours at 37° the autolyzed suspension is centrifuged for 20 minutes at 20,000 g . The extract (90 ml) is kept frozen overnight without loss of activity and is diluted with 117 ml of cold water for the subsequent procedures. The crystalline enzyme catalyzes the reversible decarboxylation of D-gluconate 6-phosphate to D-ribulose 5-phosphate and CO 2 . No evidence has been found for the existence of separate enzymatic activities catalyzing the oxidative and the decarboxylative step. The crystalline enzyme preparation contains no detectable D-glucose 6-phosphate dehydrogenase activity and is also completely free of D-ribose 5-phosphate isomerase and D-xylulose 5-phosphate 3-epimerase.

Fructose 1,6-diphosphatase from rabbit liver XIII. The number of Mn++ binding sites measured with 54Mn++

Abstract The requirement of rabbit liver FDPase for a divalent cation for catalytic activity has now been correlated with the binding of 54 Mn ++ in gel filtration studies. The enzyme binds four equivalents of 54 Mn ++ at neutral pH and four additional equivalents above pH 8.5. Only the binding of the first set of four equivalents is required for catalytic activity, and the pK of binding is approximately 7. The results provide additional support for the 4-subunit model for the enzyme.

Phosphorylation of actin and removal of its inhibitory activity on pancreatic DNAase I by liver plasma membranes

It was recently reported that the 1: 1 actinDNAase I complex is dissociated by 5’-nucleotidase and the DNAase activity is restored [ 13; the same result is obtained by incubating the actin-DNAase I complex with liver plasma membranes [2]. The a~umption, in this case, is that actin is bound to the 5’-nucleotida~ embedded in the plasma membr~es and DNAase I is released free in the solution. We have confirmed the results of these experiments and describe here a new, independent mechanism of protection of DNAase I against the inactivation by Gactin. Both G-actin and F-actin, when incubated with liver plasma membranes, are phosphorylated and lose their Inhibitory activity on DNAase I. The inactivated G-actin does not polymerize. The inactivated F-&in still activates myosin.

Chapter IV - Hexose-Monophosphate Oxidation

Publisher Summary This chapter describes the hexose-monophosphate oxidation pathway ribose 5-phosphate, which is the most important structural material generated during the operation of the cycle, as it is one of the building blocks for nucleic acid synthesis, in all organisms. Information on the relative contribution of the glycolytic and of the oxidative pathway in the breakdown of glucose 6-phosphate has been sought in experiments carried out with appropriately labeled glucose. One of the procedures is based on the evaluation of the ratio of 14 CO 2 , formed from [6- 14 C]-glucose and from [l- 14 C] glucose. However, the rate of the hexose mono-phosphate oxidation is linked to the rate of all the processes that makes use of NADPH.

G-actin modified by plasma membrane interaction polymerizes only in the presence of filamentous myosin

In non-muscle cells actin can occur in different forms depending on the immediate and local needs of the cell. This suggests the presence of regulatory mechanisms capable to allow an easy interconversion between polymerization states of actin ranging from functional microfilaments to oligomeric or even monomeric actin. We describe here a potential regulatory mechanism mediated by the interaction of G-actin with plasma membranes. After the interaction G-actin is modified insuch a way that it does not polymerize in the presence of 2 mM MgCl, but only in the presence ‘of myosin with the formation of contractile units.

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.

What is the diameter of the actin filament

The limits of the most recent models of the actin filament are discussed. These model are generated without taking into account the effect of protein osmotic pressure and, in general, of the solvent conditions. As a result they do not provide a bona fide representation of the actin filament in vivo. A new ‘fluttering wing’ model is proposed which predicts that orientation of the monomers, intermonomer contacts and diameter of the actin filament are sensitive to protein osmotic pressure and to interaction with regulatory proteins.

The interaction of histone and protamine with actin. Possible involvement in the formation of the mitotic spindle.

Abstract At low ionic strength 1–2 μM protamine or 1–2 μM H1 histone induce the nucleation of G-ATP actin. At high ionic strength 1–2 μM protamine or 0.1 to 0.2 μM H1 histone accelerate by 3 to 4 times the rate of polymerisation of G-ATP actin. It is suggested that histone may trigger the formation of actin fibers running from the kinetochore of the chromosome to the pole of the mitotic spindle.

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.