Antonio Sillero

Isoelectric points of proteins: theoretical determination

Three methods for calculating the isoelectric points (pI) of proteins, provided that their amino acid compositions are known, are described. The comprehensive and abridged procedures involve solutions of polynomial equations of different degrees depending on whether pK values of the specific acid-base residues or the means of some of those values, respectively, are adopted. In the simplified procedure, approximate pI values of proteins can be determined easily with the help of calculated values, included in this paper, related to the amino acid composition of proteins.

Diadenosine tetraphosphate activates cytosol 5′-nucleotidase

The rate of hydrolysis of IMP (0.5 mM) by cytosol 5-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5-nucleotidase from rat liver.

Dinucleosidetriphosphatase from rat brain.

Rat brain P1,P3-bis(5-adenosyl)-triphosphate adenylohydrolase (dinucleosidetriphosphatase, EC 3.6.1.29) was purified 1000-fold. The enzyme hydrolyzed diadenosine and diguanosine triphosphates (Km values 14 and 40 microM, and relative V 100 and 40, respectively) to the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was hydrolyzed at a residual rate. Diadenosine di and tetraphosphates, NAD+, and artificial phosphodiesterase substrates were not hydrolyzed. Bivalent cations [Mg(II), Mn(II) or Ca(II)] were required for activity, but Zn(II) was a competitive inhibitor (Ki = 5 microM). The optimum pH value was about 7.5. A molecular mass of 34 kdalton (gel filtration) and an isoelectric point of 5.5 (chromatofocusing) were found.

Diadenosine tetraphosphate activates AMP deaminase from rat muscle

Diadenosine tetraphosphate, Ap4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap4A appeared to induce Michaelian kinetic behavior. The activation by Ap4A was not dependent on the presence of either MgCl2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.

Dinucleosidetetraphosphatase inhibition by Zn(II)

Almost complete inhibition of partially purified dinucleoside-tetraphosphatase (EC 3. 6. 1. 17) was observed with 5 microM Zn(II). The inhibition was reversed by EDTA and was time dependent, reaching a maximum after 5 min of incubation at 37 degrees C. Zn(II) behaved as a non-competitive inhibitor of the reaction, leaving unaltered the Km value for the enzyme towards diadenosine tetraphosphate. The cellular level of this compound may be directly related to the Zn(II) content since, besides the inhibition here described, Zn(II) has been reported by others to be an activator of the synthesis of diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-t RNA synthetases.

Cytosol 5'-nucleotidase from Artemia embryos: purification and properties

Cytosol 5-nucleotidase (EC 3.1.3.5) has been purified near homogeneity from Artemia embryos. The enzyme cleaves preferentially IMP and GMP, and to a lesser extent other 5-mononucleotides. The substrate-velocity plot was hyperbolic with GMP and sigmoidal with AMP. The hydrolysis of GMP is stimulated both by ATP and beta, gamma-methyleneadenosine 5-triphosphate with the same activation constant of around 0.6 mM. Both nucleotides decreased S0.5 without affecting V. The molecular mass of the native purified enzyme was 165 kDa, and one major band of 42 kDa was detected after sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Occurrence of dinucleosidetriphosphatase in the cytosol and particulate fractions from rat liver

Dinucleosidetriphosphatase (EC 3.6.1.29) is present in both the 37,000 g rat liver supernatant and precipitate (50 mU/g each fraction). These two activities show matching molecular weights, isoelectric points, substrate specificities, Km values, bivalent cation requirements and inhibition by zinc (II). The particulate triphosphatase and a residual dinucleosidetetraphosphatase (EC 3.6.1.17) are solubilized by freeze-thawing or by Triton X-100. Detergent treatment also extracts an unspecific phosphodiesterase I activity (EC 3.1.4.1) which also splits dinucleoside polyphosphates. The above findings suggest the occurrence of cytosolic and particulate degradative pathways for dinucleoside polyphosphates.

Presence of diguanosine tri, tetra-, and pentaphosphates in commercial samples of GTP and guanosine 5'-tetraphosphate.

Commercial samples of GTP and guanosine 5-tetraphosphate were analyzed, with or without previous treatment with alkaline phosphatase, by high-pressure liquid chromatography on a Hypersil ODS column. They showed the presence of diguanosine 5,5-Pl,Pn-tri, tetra-, and pentaphosphates in varying amounts depending on the sample, but usually in proportions of around 0.3%.

IMP dehydrogenase from Artemia embryos: Molecular forms, purification and properties

Abstract 1. 1. IMP dehydrogenase (EC 1.2.1.14) has been purified near homogeneity from Artemia embryos. 2. 2. The K m values for IMP and NAD + were 15 and 200 μM, respectively. 3. 3. GMP, XMP, GTP, guanosine 5′-tetraphosphate and diguanosine tetraphosphate (Gp 4 G) were competitive inhibitors of the reaction towards IMP with K i values of 140, 180, 175, 120 and 87 μM, respectively. 4. 4. The enzyme from the 27,000 g supernatant can occur in a number of oligomeric forms (450, 375, 260 or 220 kDa) depending on the ionic strength of the medium. 5. 5. Upon precipitation with ammonium sulphate (0.3–0.4 saturation) the enzyme aggregates forming complexes of more than 1000 kDa.

Presence of cytidine 5′-tetraphosphate in commercial samples of cytidine 5′-triphosphate

A contaminant compound has been isolated from commercial samples of CTP by ion-exchange chromatography on a Dowex-1 column. It has been characterized as cytidine 5-tetraphosphate from its ultraviolet spectrum, labile and total phosphate content, and periodate consumption. It is present in proportions from 0.3 to 3.9%, apparently regardless of the method of preparation, age of sample, or commericial source of CTP.