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A Di Donato

Publications and source records attributed to A Di Donato.

18 recordsLinked to original sources
PubMed1992

The dual-mode quaternary structure of seminal RNase.

Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.

Animals↗
PubMed1992

Radiation hybrids for mapping and cloning DNA sequences of distal 16p.

Three hundred fifteen radiation hybrids (RH) were isolated using a monochromosomal cell hybrid containing chromosome 16 only. A panel of 18 RH, which showed breakpoints among four markers (3.15, 26.6, 3'HVR, and 5'HVR) mapping in the distal portion of 16p, were selected and characterized for the retention of nine additional DNA sequences already localized in this region, and for one centromeric sequence. One or more breakpoints were identified in nine of the 12 intervals defined by the 13 single-copy sequences used. This panel of RH represents a tool for the construction of a detailed physical map of the distal part of 16p and for cloning sequences located in the proximity of disease genes. Three inter-Alu DNA sequences, amplified from one of these RH containing the autosomal dominant polycystic kidney disease (PKD1) gene, were cloned and mapped in the panel. Sequencing of the ends of one of three clones showed a (CAAA)n repeat, which revealed a two-allele polymorphism.

Base Sequence↗
PubMed1992

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.

Amino Acid Sequence↗
PubMed1991

Seminal RNase: a unique member of the ribonuclease superfamily.

The RNase found in bull semen, although a member of the mammalian superfamily of ribonucleases, possesses some unusual properties. Besides its unique structure and enzymic properties, it displays antispermatogenic, antitumor and immunosuppressive activities. Seminal RNase belongs to an interesting group of RNases, the RISBASES (RIbonucleases with Special, i.e. non catalytic, Biological Actions) other members of which include angiogenin, selectively neurotoxic RNases, a lectin and the self-incompatibility factors from a flowering plant.

Animals↗
PubMed1989

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase.

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.

Bacillus↗
PubMed1989

A calorimetric approach to the study of the interactions of cytidine-3'-phosphate with bovine seminal ribonuclease.

A calorimetric study at 25 degrees C is reported on the interaction between allosteric bovine seminal ribonuclease and cytidine-3'-phosphate. The results are compared with those obtained under identical experimental conditions for the interaction of pancreatic ribonuclease A and the same nucleotide. The analysis of the data provides evidence that the binding sites of seminal ribonuclease for cytidine-3'-phosphate are not equivalent, in agreement with previous equilibrium dialysis studies. A model with two sites with different affinities toward the nucleotide, the site with higher affinity resembling the binding site of pancreatic ribonuclease, is proposed. The values calculated for the thermodynamic parameters provide an insight of the forces involved in the interaction of the two enzymes with the nucleotide.

Animals↗
PubMed1989

Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.

Amino Acids↗
PubMed1989

H-ras mutants lacking the epitope for the neutralizing monoclonal antibody Y13-259 show decreased biological activity and are deficient in GTPase-activating protein interaction.

We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Science 240:518-521, 1988). We showed that GAP had no effect on the in vitro GTPase activity of the deletion mutants of the normal p21 protein. Since similar deletions in mutants with activating lesions at position 12 or 59 or both showed decreased transforming activity, our results suggest that the recognition site for Y13-259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP and that this interaction is critical for biological activity of ras proteins.

Animals↗
PubMed1988

Co-operativity in seminal ribonuclease function. Kinetic studies.

Kinetic studies with substrates of the hydrolytic rate-limiting reaction step revealed that the non-hyperbolic kinetics of bovine seminal RNAse may not be ascribed to microheterogeneity of the enzyme or to hysteretic effects. The substrate saturation curves with intermediate plateau and the activating and inhibiting effects of the reaction product, respectively at low and high concentrations, are explained in terms of mixed co-operativity, with binding at subsites that is a prerequisite for full activity of the enzyme. A model is proposed that is supported also by the results of binding studies.

Animals↗
PubMed1987

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin.

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding, Competitive↗
PubMed1987

Increased adenylate cyclase activity in rat thyroid epithelial cells expressing viral ras genes.

The activity of the adenylate cyclase catalytic subunit is higher in Harvey and Kirsten Murine Sarcoma Viruses-infected thyroid epithelial cells than in uninfected control cells either in the presence of Mg2+ alone or following stimulation by Mn2+ or forskolin. The higher activity is associated with an increased cAMP cellular content. The Gpp(NH)p and F- anion are more effective positive modulators in the control than in the virus infected cells: these results exclude therefore that the ras p21 proteins can act as the G-protein alpha-subunit and suggest that they negatively interfere with the G-protein modulation of the adenylate cyclase system.

Adenylyl Cyclases↗
PubMed1986

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts.

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.

Adenosine Triphosphate↗
PubMed1986

Do porins inhibit the macrophage phagocyting activity by stimulating the adenylate cyclase?

Porins interact with macrophage membranes and inhibit their phagocyting activity. We have tested the porin effect on a biologically relevant membrane-bound enzymic activity, the adenylate cyclase system, which appears to be stimulated both in the presence of Mn2+ and Mg2+ or Mg2+ + Gpp(NH)p. Moreover, for mice macrophages incubated in the presence of porins, there is an increase in the intracellular cAMP content after 5 min of incubation, with a maximum after 15 min of incubation. The results shown suggest that the porin effects on the adenylate cyclase can represent the molecular basis of the porin-dependent inhibition of the macrophages phagocytosis. Our point of view, which proposes a cAMP role in inhibiting the phagocyting activity in macrophages, is supported also by the results of the experiments carried out in the presence of both dibutyryl-cAMP or aminophylline. The phagocyting activity is inhibited in all cases and independently of the bacteria to be phagocyted.

Adenylyl Cyclases↗
PubMed1985

The effects of lidocaine and procainamide on the sarcolemmal membrane high affinity cyclic AMP phosphodiesterase of rat myocardium.

The incubation of rat myocardial sarcolemmal membranes with 10(-4)M lidocaine or procainamide results in a decreased fluorescence polarization of the extrinsic probes (diphenylhexatriene and 12-anthroylstearate) and in the stimulation of the membrane-bound high affinity cyclic AMP phosphodiesterase activity (PDE). Lidocaine or procainamide do not contrast, but facilitate the aminophylline inhibitory action on the PDE activity, without modifying the basic molecular mechanism of the inhibitory action. The amplitudes of the effects described are inversely correlated to the temperature, being greater at the lower tested temperature (25 degrees greater than 30 degrees greater than 37 degrees C).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

[Preliminary data on activation of the enzyme of the metabolism of phospholipid bases in various areas of the brain].

The scope of this work was to study the effect of imydazole on base-exchange enzymic system in selected cerebral areas. We have previously demonstrated that imydazole was an activator of phosphatidylserine synthesis in slices of caudate nucleus. This effect lacked in the omogenate we had supposed that this activation by imydazole was not directed on base-exchange enzymic system, but was induced by decrease of cellular cyclic nucleotides amount. Therefore we have investigated the effect of dibutirril-AMPciclic in selected cerebral areas. In addition it was useful to state if activation by imydazole was area-specific or not.

Animals↗
PubMedDate not supplied

Mapping of the human COL5A1 gene to chromosome 9q34.3.

A 353-bp region encoding for the NH2 terminus of the noncollagenic part of the alpha 1(V) chain was amplified by the polymerase chain reaction (PCR), subcloned and sequenced. The subcloned PCR product (pGC1) presented the same nucleotide sequence as the original fragment from the published sequence of COL5A1. In situ hybridization, using pGC1 as a probe, mapped the COL5A1 gene to chromosome 9q34.3. This assignment shows that COL5A1 is not synthetic with COL5A2, which is localized together with other collagen genes on chromosome 2.

Base Sequence↗
PubMedDate not supplied

Activation of phosphatidylserine synthesis: a possible mechanism of regulation of the base-exchange enzymic system.

The possible relationship between phosphatidyl serine synthesis by base-exchange and nervous activity has been investigated in the rat caudate nucleus. The rate of incorporation of L-serine into the phosphatidyl serine of slices from caudate nucleus is not affected by dopamine nor is it affected by the addition to dopamine of a cyclic phosphodiesterase inhibitor which would increase the endogenous cyclic-AMP levels. However, imidazole, a phosphodiesterase activator, clearly stimulates by more than 100% the phosphatidyl serine synthesis in the slices. The activation is not due to interaction at the catalytic site(s) of the base-exchange system, since it is neither observed in homogenates of caudate nucleus nor in cerebral microsomes at various pH values.

3',5'-Cyclic-AMP Phosphodiesterases↗