PubMed Health⌕ Search

Biomedical subjects

C M Cuchillo

Publications and source records attributed to C M Cuchillo.

At least 37 records · Page 2Linked to original sources
PubMed1993

The role of 2',3'-cyclic phosphodiesters in the bovine pancreatic ribonuclease A catalysed cleavage of RNA: intermediates or products?

There is a considerable degree of ambiguity in the literature regarding the role of the 2',3'-cyclic phosphodiesters formed during the reaction of RNA cleavage catalysed by ribonuclease. Usually the reaction is considered to take place in two steps: in the first step there is a transphosphorylation of the RNA 3',5'-phosphodiester bond broken yielding a 2',3'-cyclic phosphodiester which in the second step is hydrolysed to a 3'-nucleotide. Although in many occasions, either explicitly or implicitly, the reaction is treated as taking place sequentially, this is not the case as it has been shown that the 2',3'-phosphodiesters are actually released to the medium as true products of the reaction and that no hydrolysis of these cyclic compounds takes place until all the susceptible 3',5'-phosphodiester bonds have been cyclised. Comparison of the hydrolysis and alcoholysis of the 2',3'-phosphodiesters catalysed by RNase A indicates that the hydrolysis reaction has to be considered formally as a special case of the transphosphorylation back reaction in which the R group of the R-OH substrate is just H. It is thus concluded that the 2',3'-cyclic phosphodiesters formed in the ribonuclease A reaction are true products of the transphosphorylation reaction and not intermediates as usually considered.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1991

Structure and function of ribonuclease A binding subsites.

Ribonuclease A binds nucleic acids through multiple electrostatic interactions between the phosphates of the polynucleotide and the positive groups (side chains of lysines and arginines) of the protein subsites. The bases only play a significant role in the binding at the active site. The active centre p1R1B1 sites determine the specificity of the catalytic cleavage. The phosphate-binding subsites p2 (Lys-7 and Arg-10), p1 (Lys-41, His-12 and His-119) and p0 (Lys-66) are essential for an effective catalysis and are conserved in all mammalian pancreatic ribonucleases. Additional phosphate-binding subsites confer further catalytic efficiency, probably by avoiding non-productive binding. The minimum chain size for optimum catalysis is probably longer than six or seven nucleotides. The full occupancy of binding sites by the long chain polynucleotides would explain the preference of the enzyme for these substrates. The multiplicity of binding subsites is responsible for the helix-destabilizing activity of ribonuclease A. Its capacity for destroying the secondary structure of single-stranded nucleic acids may be of importance for the complete hydrolysis of RNA in the digestive tract. A large variety of proteins, with very different structures and functions, interact with nucleic acids. An analysis of their binding properties shows that there is no general model for protein-nucleic acid interaction. However, the vast amount of work on the ribonuclease A binding subsites should serve as a model for the study of the binding properties of many other proteins that recognize nucleic acids.

Animals↗
PubMed1990

Chemical modification by pyridoxal 5'-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A.

Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.

Animals↗
PubMed1990

The tryptic activation pathway of monomeric procarboxypeptidase A.

Procarboxypeptidases are the remaining major digestive zymogens the activation process of which remains unsolved. Here it is shown that in the tryptic activation of monomeric procarboxypeptidase A from porcine pancreas, the generation of carboxypeptidase A (CPA) activity parallels the limited proteolysis of the 94-residue activation segment. This degradation proceeds from the COOH-terminal end of the molecule, and CPA itself makes an important and unexpected contribution by excising the COOH-terminal arginine residue of the released primary activation fragment. Successive cleavages at some of the peptide bonds of the activation segment nearest to the COOH terminus were found to be of prime importance in eliciting CPA activity, particularly those involving the carbonyl groups of Arg94 and Gly93 which were first cleaved. It is also shown that the rate of activation does not depend directly upon the generation of CPA-alpha and its conversion to CPA-beta.

Amino Acid Sequence↗
PubMed1989

The separation of pancreatic procarboxypeptidases by high-performance liquid chromatography and chromatofocusing.

Different experimental conditions and chromatographic supports have been selected for the most efficient and rapid purification of procarboxypeptidases from porcine and human pancreas by different high-performance liquid chromatography (HPLC) variants (anion exchange, reversed phase and gel filtration). Anion-exchange chromatography was found to be the most capable and permitted the isolation, in a single step, of three different porcine procarboxypeptidases (2A + 1B forms) and five different human procarboxypeptidases (2B + 3A forms) in a native and pure state from whole pancreas extracts. Other pancreatic proproteases are also cleanly isolated in the same step. Reversed-phase chromatography under mild conditions separated porcine or human procarboxypeptidases A from other pancreatic proteins in a very short time but was unable further to subfractionate the same proteins. The sequential use of gel filtration (or anion-exchange) and reversed-phase HPLC chromatography permitted, in a simple way, the isolation and dissociation of the strongly bound components of the binary complexes between procarboxypeptidases A and proproteinase E in either porcine or human pancreas extracts. Chromatofocusing on a fast protein liquid chromatographic support was also found to be a very efficient technique, showing a slightly lower capability to separate procarboxypeptidases than anion-exchange HPLC though in a much shorter time and in larger quantities.

Animals↗
PubMed1989

Kinetic analysis of the carboxypeptidase A hydrolysis of oligopeptides by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic (HPLC)-based method was developed to follow the time course of the hydrolytic action of pancreatic carboxypeptidase A on oligopeptide substrates of the general formula benzoyl(glycyl)n-L-Phe, n being in the range 1-5. The proposed procedure is sensitive at the nanomolar level of substrate, but also allows the kinetics of substrate hydrolysis to be investigated over a wide range of concentrations. The prior quenching of the carboxypeptidase activity and the use of isocratic conditions for the rapid separation of the substrates and their products (in less than 8 min for the slowest one), allow the automation of the procedure, which could become an easy and versatile alternative to the commonly used spectrophotometric methods. A comparative evaluation of the use of a spectrophotometric method to measure carboxypeptidase A activity on the same substrates indicated that the latter is not valid for long oligopeptides (n greater than or equal to 2) at concentrations higher than 5 mM owing to an interference of a physico-chemical nature. For benzoylglycyl-L-Phe (n = 1), the apparent kinetic parameters were calculated by means of the HPLC method and by a well established spectrophotometric procedure, and both yielded similar values.

Animals↗
PubMed1989

Interference of the carbohydrate moiety in coomassie brilliant blue R-250 protein staining.

The binding of Coomassie Brilliant Blue R-250 to several species of bovine pancreatic ribonuclease is affected by the presence of a carbohydrate moiety in the enzyme molecule. Enzymic deglycosylation of several chromatographic fractions of ribonuclease, which have different degrees of glycosylation, results in increased staining by Coomassie Brilliant Blue R-250. Ovalbumin and other glycoproteins tested show similar behavior. The results indicate that carbohydrate moieties may represent a common hindrance to the binding of Coomassie Brilliant Blue dyes to glycoproteins.

Animals↗
PubMed1989

Chemical and computer graphics studies on the topography of the ribonuclease A active site cleft. A model of the enzyme-pentanucleotide substrate complex.

The affinity labelling of bovine pancreatic ribonuclease A with 6-chloropurine 5' ribonucleotide allowed us to postulate the existence of a new phosphate-binding subsite, P2, adjacent to the main purine-binding subsite. The study of this reaction in greater detail together with the study of a complex of the enzyme with the pentanucleotide pApUpApApG by means of model building and computer graphics indicate that at least five phosphate groups of the RNA molecule can interact with five positive regions of the enzyme. In each one a lysine residue is present: Lys-104, -66, -41, -7 and -37 appear sequentially in the 5'----3' direction. The distance between each lysine is 0.7-0.8 nm, the same distance as that found between the phosphate groups on the RNA molecule. The study also enabled many amino acid residues of the enzyme to be described as forming part of, or being near, the different binding subsites.

Binding Sites↗
PubMed1989

1H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A.

The reaction of ribonuclease A with either 6-chloropurine riboside 5'-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the alpha-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of 1H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3'AMP is similar to that described for RNase A as the pK2 of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK2 values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK2 of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.

Adenosine Monophosphate↗
PubMed1988

Analysis of the conformation and ligand-binding properties of the activation segment of pig procarboxypeptidase A.

The isolated activation segment of pig procarboxypeptidase A binds two Tb3+ ions in a strong and specific way. In contrast, the binding of Ca2+, Cd2+ and Mg2+ is weak. The binding of Tb3+ increases the resistance of the isolated activation segment against proteolysis and competes for the binding of the carbocyanine dye Stains-All. This dye forms complexes with the activation segment showing spectral properties similar to those observed with EF-hand structures. The presented results support a previous hypothesis on the existence of two regions in the activation segment of pancreatic procarboxypeptidases structurally related to Ca2+-binding domains of the EF-hand protein family.

Animals↗
PubMed1988

Nucleotide binding and affinity labelling support the existence of the phosphate-binding subsite p2 in bovine pancreatic ribonuclease A.

When the reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate was carried out in the presence of several natural mononucleotides, a decrease of 25-75% was found in the amount of the reaction product derivative II (the main product of the reaction which has the nucleotide label at the alpha-NH2 group of Lys-1). The efficiency of inhibition followed the order 3'-AMP greater than 5'CMP approximately equal to 5'AMP greater than 3'CMP. Previous studies indicate that this order reflects the extent of occupancy of p2, a phosphate-binding subsite adjacent to the catalytic centre. This finding suggests that derivative II is the result of affinity labelling and that the phosphate group of the halogenated nucleotide binds to p2 before the reaction takes place. The dissociation constants and stoichiometry of the interaction between native enzyme, derivative II and derivative E (homologous to derivative II, but labelled with a nucleoside instead of a nucleotide) with 3'AMP and 5'AMP at several pH values were also determined. Although in general one strong binding site was found, no strong binding occurs between 3'AMP and derivative II. It is concluded that the phosphate of the label occupies the same site p2, as the phosphate of 3'AMP. Finally, the pH dependence for the binding of 3'AMP and 5'AMP to RNAase A indicates that they bind to different protein groups. The results presented support the structure of the active site of ribonuclease A postulated previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571-579).

Adenosine Monophosphate↗
PubMed1988

H-n.m.r. studies on the specificity of the interaction between bovine pancreatic ribonuclease A and dideoxynucleoside monophosphates.

The titration curves of the C-2 histidine protons of bovine pancreatic ribonuclease A in the presence of several dideoxynucleoside monophosphates (dNpdN) were studied by means of proton nuclear magnetic resonance at 270 MHz in order to obtain information on the ligand--RNase A interaction. The changes in the chemical shift and pKs of the C-2 proton resonances of His-12, -48, -119 in the complexes RNase A--dNpdN were smaller than those previously found when the enzyme interacted with mononucleotides. The pK2 of His-12 was not affected by the interaction of the enzyme with these ligands, whereas, the perturbation of the pK2 of His-119 was clearly dependent on the nature of the ligand. If there is a pyrimidine nucleoside at the 3' side of the dideoxynucleoside monophosphates, as in TpdA and TpT, an enhancement due to the well known interaction of the phosphate in p1, the catalytic site, was found. However, when there is a purine nucleoside, as in dApT and dApdA, a decrease in the pK2 value was observed and we propose that in such cases the phosphate group interacts in a secondary phosphate binding site, p2. The results obtained suggest the existence of different specific interactions depending on the structure of the dideoxynucleoside monophosphate studied.

Deoxyribonucleotides↗
PubMed1986

Modification of bovine pancreatic ribonuclease A with 6-chloropurine riboside.

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine riboside was studied to obtain information about the role of the purine nucleoside moiety of the ribonucleic acid in the enzyme-substrate interaction. The residues involved in the reaction were identified, after performic acid oxidation and trypsin digestion, by reverse-phase HPLC peptide mapping. The labeled peptides were detected by following the absorbance at 254 nm, and amino acid analyses of these peptides showed that the reaction had taken place with the amino groups of Lys-1, -37, -41, and -91. The specificity of the reaction was unaffected by changing the ligand:protein molar ratio. Partial separation of the reaction products was accomplished by means of chromatography on CM-Sepharose: four labeled fractions corresponding to mono- and bisubstituted derivatives were found. One of the monosubstituted fractions (fraction E) contained a homogeneous protein with the nucleoside bound to the alpha-amino group of Lys-1 whereas the other (fraction D) was a mixture of derivatives labeled in the epsilon-amino group of Lys-1, -37, -41, and -91. Kinetic studies of these two monosubstituted fractions were performed with cytidine 2',3'-phosphate and ribonucleic acid as substrates. These derivatives showed a noncompetitive inhibition-like behavior with respect to RNase A. Results support the existence of several RNase A regions with affinity for purine nucleosides.

Animals↗
PubMed1985

Urea-gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products.

Monomeric procarboxypeptidase A (PCPA) and isolated proproteinase E (PPE), both from pig pancreas, were shown by means of electrophoresis on transverse urea gradients (0-9 M) to form a very stable complex, identical to their natural binary complex. Although the complex is maintained by the interaction of both active regions, the activation segment of PCPA participates directly in the binding. Procarboxypeptidase B (PCPB) also associates with PPE, but in this case the complex shows low stability. In contrast with carboxypeptidases A that strongly bind to their corresponding severed activation segments, no interaction was observed between carboxypeptidase B and its severed activation segment. The above results give some insight into several characteristics of the structure and activation properties of pancreatic PCPA and PCPB.

Animals↗
PubMed1985

Different kinetic patterns in the alpha-chymotrypsin-catalysed hydrolysis of synthetic ester substrates.

The reaction of alpha-chymotrypsin with AcTyr-OEt and with AcTrp-OEt at pH 7.0 and 7.8 was studied over a wide range of substrate concentrations. The reaction with AcTyr-OEt at pH 7.8 was shown to be non-hyperbolic using a variety of criteria whereas those at pH 7.0 with the same substrate and at both pH values with AcTrp-OEt were hyperbolic. The non-hyperbolicity of the reaction with AcTyr-OEt at pH 7.8 followed a pattern of negative cooperativity with a Hill coefficient for the high substrate concentration range of 0.48. Although other explanations are possible, the pH dependence of the reaction with AcTyr-OEt could be related to the slow transition of the two known forms of the enzyme.

Carboxylic Acids↗
PubMed1985

Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A.

A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods. Undegraded and homogeneous carboxypeptidase A1 and A2 species (peptidyl-L-amino acid hydrolase, EC 3.4.17.1), in monomeric forms with high specific activity, are also obtained in high yield by controlled trypsin activation of either of the pro-(carboxypeptidases A) followed by chromatography on DEAE-Sepharose at pH 5.8 under dissociating conditions (7 M-urea).

Animals↗
PubMed1984

The properties of the interaction between bovine pancreatic ribonuclease a and mononucleotides supports the existence of several binding sub-sites in the enzyme.

The binding of 5'AMP, 5'GMP, 5'CMP, 3'CMP and Cl6RMP to RNAase A was studied by means of the gel filtration technique. It was found that only one molecule of 3'CMP binds strongly to the enzyme although a very unspecific binding is also present. The interaction of 5'AMP and 5'GMP with the enzyme shows one strong binding site and several weak binding sites, whereas two molecules of 5'CMP bind to RNAase A with equal strength. Cl6RMP shows an anomalous behaviour as both split peaks and troughs are found in the chromatogram. The Ka values for 3'CMP and the strong binding site of 5'AMP and 5'GMP are very similar whereas that for the two binding sites of 5'CMP is smaller (about 2.2 X 10(-4)M-1 and 0.5 X 10(-4)M-1, respectively at pH 5.5, I = 0.01 and 25 degrees C). The results are in general agreement with the known multiplicity of ligand-binding subsites in RNAase A.

Animals↗