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C M Cuchillo

Publications and source records attributed to C M Cuchillo.

At least 55 records · Page 3Linked to original sources
PubMed1983

Pro-(carboxypeptidases A) from whole pig pancreas. Their mass, size, shape and solvation.

Sedimentation analysis and light-scattering measurements were made with the two forms of pig pancreas pro-(carboxypeptidase A), in order to determine some of their physical properties. The following values were found (the first value applies to the binary complex and the second one to the monomer). The A 1%/280.1 cm values were 19.9 +/- 0.3 and 16.3 +/- 0.3. The partial specific volumes v -0 were 0.707 +/- 0.016 cm3/g and 0.714 +/- 0.015 cm3/g. The sedimentation coefficients S 0/20,w were 4.90 +/- 0.15S and 3.75 +/- 0.15 S. The diffusion coefficients D 0/20,w were (5.8 +/- 0.1) X 10(-7) cm2/s and (6.95 +/- 0.15) X 10(-7) cm2/s. From these data the following values were calculated. Relative molecular masses Mr were 71 000 +/- 4000 and 46 000 +/- 3000. The frictional ratios f/fmin. were 1.37 +/- 0.06 and 1.31 +/- 0.07; assuming a value for the solvation of the molecules (delta = 0.5 g/g) the asymmetry values range from 3 to 5 for the binary complex and from 2 to 4 for the monomer. The Mr values found in the present work coincide with those found by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate [Martínez, Avilés, SanSegundo & Cuchillo (1981) Biochem. J. 197, 141-147]. Therefore the low values obtained by those authors when using gel-filtration chromatography must be the result of the interaction of the zymogens with the gel matrix, as the asymmetry is too small to justify the large discrepancies found.

Animals↗
PubMed1983

Affinity chromatography study of the interaction of ribonucleotides with bovine pancreatic ribonuclease covalently bound to Sepharose 4B.

The preparation of immobilized bovine pancreatic ribonuclease by covalent attachment to Sepharose 4B, with and without a spacer arm, is described. The coupling reaction was carried out at two different pH values, 8.5 and 10.5, and the different kinetic properties shown by the resulting preparations probably reflect the influence of the particular amino acid side-chains involved in the covalent coupling of the enzyme to the insoluble matrix. The strength of binding of mononucleotides, at 4 degrees C, as deduced from the salt concentration at which they are eluted from an immobilized RNAase column, follows the order 5'-GMP greater than 5'-AMP greater than 3'-UMP greater than 3'-CMP. When binary mixtures of a 3'-pyrimidine nucleotide and a 5'-purine nucleotide are chromatographed jointly, a co-operative effect is found and the elution of either or both ligands is retarded. This behaviour can be explained in terms of the preferential binding of each kind of nucleotide to different sub-sites in the enzyme. The stoichiometry and association constant for 3'-CMP and 5'-AMP at pH 7.0 were also determined.

Animals↗
PubMed1982

The activation segment of procarboxypeptidase A from porcine pancreas constitutes a folded structural domain.

The controlled action of trypsin on porcine pancreatic procarboxypeptidase A releases a large activation peptide which contains the activation segment of the proenzyme. Circular dichroism studies indicate that the isolated activation peptide contains a high percentage of residues in ordered secondary structures (mainly alpha-helix). This result agrees with predictions of secondary structure carried out on the published amino acid sequence of the homologous rat proenzyme. Moreover, proton magnetic resonance spectroscopy shows that the peptide adopts a thermostable tertiary structure with characteristics typical of globular proteins. The results as a whole indicate that the activation segment of porcine pancreatic procarboxypeptidase A constitutes a folded structural domain.

Animals↗
PubMed1982

The severed activation segment of porcine pancreatic procarboxypeptidase A is a powerful inhibitor of the active enzyme. Isolation and characterisation of the activation peptide.

The activation peptide of the monomeric procarboxypeptidase A from porcine pancreas was isolated by means of controlled trypsin digestion of the proenzyme followed by ion-exchange chromatography under dissociating conditions (7 M urea). The molecular weight of the isolated peptide was estimated to be around 11500-12000 (corresponding to approx. 100-103 residues) as judged by SDS electrophoresis and amino acid analysis, a figure that agrees with the differences between the corresponding values for procarboxypeptidase A and carboxypeptidase A (peptidyl-L-amino acid hydrolase, EC 3.4.17.1). The activation peptide has a high content of hydrophobic and acidic amino acids, and lacks cysteine. A remarkable feature is the strong competitive inhibitory action of the peptide on both porcine and bovine pancreatic carboxypeptidase A activity, with a Ki in the nanomolar range, and its null ability to inhibit porcine pancreatic carboxypeptidase B (EC 3.4.17.2). The above properties, and the fact that the peptide has the same N-terminal residue (lysine) as the parent procarboxypeptidase A, suggest that the isolated peptide contains most (if not all) of the activation segment of the proenzyme.

Amino Acids↗
PubMed1982

Evidence on the existence of a purine ligand induced conformational change in the active site of bovine pancreatic ribonuclease A studied by proton nuclear magnetic resonance spectroscopy.

The titration curves of the C-2 histidine protons of RNase A and of derivative II--a covalent derivative obtained by reaction of the enzyme with the halogenated nucleotide 9-beta-D-ribofuranosyl-6-chloropurine 5'-phosphate--in the presence of a number of purine nucleosides, nucleoside monophosphates, and nucleoside diphosphates were studied by means of proton nuclear magnetic resonance at 270 MHz. The examination of the perturbations found on the chemical shifts and pKs of the C-2 protons of His-12, -48, and -119 are consistent with the following conclusions: (1) The interaction of adenosine in the primary purine binding site of the enzyme (B2R2) induces a conformational change in the active center of the enzyme [for the nomenclature of the RNase A binding subsites, see Parés et al. [Parés, X., Llorens, R., Arús, C., & Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579]]. (2) The phosphate moiety of the ligands, independently of its position, probably acts as a general carrier of the nucleotide to the active center, while the substituents of the base are the generators of the specificity of the binding and control the binding equilibrium between subsites B2R2 and B1R1. (3) There is no overlapping between the binding sites occupied by the labeling nucleotide in derivative II (B3R3p2) and the primary binding site for purine mononucleotides (B2R2p1).

Adenosine↗
PubMed1982

Isolation and re-association of the subunits from the pro-(carboxypeptidase A)-pro-(proteinase E) binary complex from pgi pancreas.

The component subunits of the pro-(carboxypeptidase A)-pro-(proteinase E) binary complex from pig pancreas were separated with a high recovery (80-95%) of their original potential activity. The isolated subunits and the reconstituted complex have properties similar to those of the corresponding natural species. The tryptic activation course of the pro-(carboxypeptidase A) subunit is substantially modified when bound to pro-(proteinase E), whereas the activation of pro-(proteinase E) is not dependent on this association.

Animals↗
PubMed1982

Influence of the structure of water on the hydrolysis of cytidine 2',3'-phosphate catalysed by bovine pancreatic ribonuclease A.

The study of the temperature dependence of the hydrolysis of cytidine 2',3'-phosphate by bovine pancreatic ribonuclease A (EC 3.1.27.5) at pH 7.0 by using the pH-stat method showed a transition at 4 degrees C [J. A. Biosca and C. M. Cuchillo (1980) Biochem. J. 189, 655-657]. The breaks found in the Van't Hoff and Arrhenius plots at pH 7.0 were confirmed in the present work by following the reaction spectrophotometrically with a stopped-flow spectrophotometer adapted to the use of sub-zero temperatures. Similar results were found at pH 5.5. In addition it was found that the discontinuity disappears when 40% ethyleneglycol is present in the reaction mixture. However, in this latter instance a discontinuity around 0 degrees C appears in the Arrhenius plot. To explore the possibility that all these effects were due to a conformational transition in the protein, thermal perturbation experiments were carried out with the enzyme. A change in the slope of the plot of delta A290 as a function of temperature was found around 6 degrees C at pH 7.0 but not at pH 5.5. The results reported here can be interpreted as due to a change in the protein structure induced by the change of the structure of water. The studies carried out in the presence of ethyleneglycol also open the way to the cryoenzymological experimentation on ribonuclease.

Animals↗
PubMed1981

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A.

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.

Animals↗
PubMed1981

Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas.

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.

Amino Acids↗
PubMed1981

Inhibition of phytohaemagglutinin-induced transformation of lymphocytes by human spleen extracts.

The presence in human spleen extracts of a substance capable of inhibiting the phytohaemagglutinin (PHA)-induced transformation of lymphocytes into lymphoblasts was demonstrated by analysing the nuclear size of cultured human lymphocytes. In this way the artefacts produced by the methods based on the uptake of 3H-thymidine were avoided. The inhibitor did not compete with PHA for the same membrane receptor-site, since adding the extract 24 h after the PHA-stimulation still produced a significant inhibition. The lack of cytotoxicity of the extract was demonstrated by the fact that cultures that lasted for six days, instead of three days, resulted in a pattern similar to that of control cultures. An attempt to partially purify the inhibitor by means of ammonium sulphate fractionation showed that the highest activity precipitated between 0.5 and 0.6 saturation although, due to the characteristics of the assay, it was difficult to assess the actual degree of purification.

Cell Nucleus↗
PubMed1980

Thermodynamic studies on the hydrolysis of cytidine 2':3'-phosphate by bovine pancreatic ribonuclease A. A possible effect of the change of the structure of water.

The temperature-dependence of the ribonuclease A-catalysed hydrolysis of cytidine 2':3'-phosphate was studied in the range of temperatures 0--40 degrees C. A break at 4 degrees C was found both in the Arrhenius and the van't Hoff plots. It is likely that the transition observed is due to the change in the structure of water.

Animals↗
PubMed1980

The reaction of bovine pancreatic ribonuclease A with 6-chloropurineriboside 5'-monophosphate. Evidence on the existence of a phosphate-binding sub-site.

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate was studied under several reaction conditions. The reaction, at pH 7.3, 40 degrees C and a nucleotide: enzyme molar ratio of 60, showed a high degree of specificity in comparison to those corresponding to the base or the nucleoside. The main derivative was isolated by means of CM-cellulose chromatography. Subtilisin cleavage of this derivative showed that the substitution had taken place in the S-peptide moiety. Tryptic digestion of the S-peptide indicated that a lysine residue had been modified. Enzymatic and physico-chemical considerations showed that the actual site of reaction was the alpha-amino group of Lys-1. The structural and kinetic properties of the derivative are consistent with the existence of a phosphate-binding sub-site near the N-terminal region of the enzyme.

Amino Acids↗
PubMed1980

Reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate. Nuclear magnetic resonance studies of the corresponding S-peptide.

The n.m.r. spectra of native S-peptide and of S-peptide II, a derivative obtained after reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate, both in D2O and in urea-d4, were obtained with a 270 MHz Fourier transform spectrometer. From these spectra it was possible to assign most of the proton resonances of the peptide and the position of the labelling group, the alpha-NH2 of Lys-1, was also deduced.

Animals↗
PubMed1978

The specificity of the interaction of bovine pancreatic ribonuclease A with natural and halogenated purine nucleotides.

The interaction between bovine pancreatic ribonuclease A (EC 3.1.4.22) and the purine nucleotides AMP, GMP, 6-chloropurine 5'-ribonucleotide and 8-bromoadenosine 5'-monophosphate was studied by u.v. difference spectroscopy. The stoicheiometry of the binding of the halogenated nucleotides to the enzyme shows a 1:1 ratio, as for the natural ones. The binding constants, Ka, for all four nucleotides at pH 5.5 were determined. They are within the same order of magnitude, though the nucleotides with a 6-amino group show a stronger interaction. The magnitude of the binding shows a reciprocal dependence on the ionic strength, which indicates an electrostatic interaction between ligand and enzyme. Finally, solvent-perturbation experiments show that all four nucleotides bind to the enzyme in a partially hydrophobic region. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule. The special synthesis and identification of 6-chloropurine 5'-ribonucleotide are discussed extensively. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule and thus the halogenated analogues are potential reagents for the affinity labelling of the purine-binding site.

Binding Sites↗