Patrick J. Cozzone

Characterization of Porcine Pancreatic Isoamylases Chemical and Physical Studies

Summary The amino acid analyses of amylases I and II are given. No significant differences between the amino acid compositions were found with the exception that porcine pancreatic amylase I contained five more aspartic acid + asparagine residues than amylase II. Both porcine α-amylases possessed amino acid composition features common to all α-amylases of known amino acid content. A common C-terminal sequence -Ile-Val-Tyr-Phe-Leu-COOH and a blocked N-terminal group were found in the isoenzymes. These results, together with gel electrophoresis and gel filtration analysis of reduced and reduced then S-carboxy-methylated (RCM) amylase, indicate a monomeric enzyme consisting of a single polypeptide chain. The molecular weight determined by four different techniques was found to be in the range 51 000-54 000.

LIMITED TRYPSINOLYSIS OF PORCINE AND EQUINE COLIPASES - SPECTROSCOPIC AND KINETIC-STUDIES

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of bile salt-inhibited lipase with emulsified triolein in the absence and in the presence of lecithin show that tryptic hydrolysis of the protein cofactor increases its affinity for the enzyme in the presence of lipid substrate. In both cases, it was found that the apparent dissociation constant of the lipase-colipase complex is decreased by one order of magnitude. Our results confirm that the biological activity of the lipase cofactor is enhanced by specific tryptic cleavage in the amino terminal region of the polypeptide and support the suggestion by Borgström et al. (Borgström, B., Wieloch, T., Erlanson-Albertsson (1981) FEBS. Lett. 108, 407-410) that the secreted form of colipase is a precursor.

Conformational dynamics of porcine pancreatic colipase: A 360 MHz proton nuclear magnetic resonance study

The present study is devoted to colipase II, one of the two main molecular forms of porcine pancreatic colipase [1,2]. This small protein has a molecular weight of 8700 and consists of a single polypeptide chain of 84 amino acid residues [3,4]. Its amino acid sequence has been determined [3] and corresponds to a rather compact structure maintained by 5 disulfide bridges [4]. The biological role of colipase is of great importance since it acts in vivo as a cofactor preventing the inhibitory effect of physiological concentrations of bile salts on the intraduodenal lipolysis of dietary triglycerides [1,5,6]. In an attempt to explain the physiological effect of colipase, binary and ternary associations of colipase, lipase and bile salts have been recently investigated under a variety of experimental conditions [7-13] . Typically, colipase has been shown to bind stoichiometrical amounts of bile salt micelles and to form a binary complex that pancreatic lipase can in turn recognize. It is likely that these binding and recognition processes involve specific conformational changes creating and/ or unveiling appropriate sites on the colipase molecule [ l l ] . The potential of high resolution n.m.r, in describing the conformational rearrangements of proteins under various perturbing factors (pH, temperature, ionic strength, ligand binding etc...) is now widely recognized. In this paper, I report and describe the first n.m.r.

360 MHz laser photo-CIDNP of porcine pancreatic colipase A: Study of the aromatic surface residues

Colipase is a protein of low molecular weight (about 11 000) found in the pancreatic secretion of mammals [ 11. Its physiological role is, (i) to allow lipase to adsorb at lipid-water interface in the presence of high concentrations of bile salts, (ii) to stabilize the enzyme in its fully active form under large variations of surface energy [2-51. The adsorption of colipase to the substrate-water interface is now accepted as the first step of the lipolytic process and it can be related to the presence of a particular domain on the surface of the coprotein [6]. Proton NMR, fluorescence and spectrophotometric studies on porcine colipase have indicated that two tyrosine and one histidine residues are located in a hydrophobic region of the protein that might constitute at least a part of the lipid binding site on colipase [7-l 11. Here we report a series of laser photo-CIDNP experiments performed on porcine colipase A [ 111. The photo-CIDNP method is based on the generation of nuclear spin polarization in a reversible photoreaction of a dye with aromatic residues [ 121. The sidechains of tyrosine, histidine and tryptophan residues can be polarized when they are accessible to the photo-excited dye [13,14]. Porcine colipase A contains 3 tyrosine, 2 histidine and 2 phenylalanine

The mechanism of purine polynucleotide hydrolysis by ribonuclease A

It is generally held that bovine pancreatic ribonuclease (RNAase) is an enzyme specific for phosphodiester bonds in which the 3’-linked nucleotide is a pyrimidine [ 1 ] . Slow hydrolysis of purine nucleotides has nevertheless been observed in ribonuclease preparations and is sometimes attributed to other contaminating nucleases [2]. In this communication, we wish to report observations made by 31P NMR which indicate that the hydrolysis of single stranded poly(A) in the presence of ribonuclease proceeds via a 2’,3’-cyclic intermediate. The kinetics of the reaction suggest that the cyclization step is enzymatic, and the hydrolysis is completed non-enzymatically by the basic solvent (pH 7.90, held constant), yielding a mixture of 2’ and 3’-AMP.

Proton NMR study at 360 MHz of porcine pancreatic colipase: Identification of aromatic resonances

Colipase is a small protein cofactor found in the pancreatic secretion of mammalians [1,2]. The biological role of colipase is of critical importance in vivo since it counteracts the inhibitory effect of physiological concentrations of bile salts on the intraduodenal lipolysis of insoluble dietary triglycerides [ 1-3] . In addition, colipase has a direct specific effect on interracial lipolysis by protecting lipase from surface denaturation in the absence of detergents [4-6] . Considering that these binding and recognition processes are likely to involve specific structural changes creating and/or unveiling appropriate sites on the colipase molecule, we have undertaken a systematic NMR study of colipase conformational properties in solution. A general interpretation of the colipase II spectrum has already been reported [7]. Colipase II is one of the two major forms of the cofactor isolated in [8] ; its covalent structure (84 residues) has been fully elucidated [9,10] and corresponds to a compact central core crosslinked by 5 disulfide bridges with two terminal tails. Two other forms of the cofactor have been obtained [11,12] and some characteristics of 3 aromatic residues of a 106 residue long form of colipase have been investigated by NMR spectroscopy [ 13 ]. A new direct purification of porcine colipase from pancreatic tissue homogenate in the presence of Triton X-100 has been achieved [ 14]. The major form (80%) thus obtained and designated as colipase A is highly stable and is similar to the one in [11,12].

Isolation and characterization of the cyanogen bromide peptides of 2 forms of porcine pancreatic amylase

Summary Porcine pancreatic amylases I and II have identical activities, molecular weight, end groups and amino acid composition (except for aspartic acid and/ or asparagine residues). In order to get more information about the structure of the 2 forms, both amylases were cleaved by cyanogen bromide and the fragments thus obtained were purified by gel filtration and analyzed. Kinetics of the cleavage indicates a complete and specific splitting at the methionine level. Successive filtrations of the peptides were performed in 30 p. cent propionic acid on appropriate columns. Nine pure peptides were thus obtained and characterized by their molecular weight, end groups and amino acid composition. The location of the 4 disulfide bridges and of the 2 free SH groups in the peptides was determined. The N and C-terminal peptides were identified, Surprisingly, no difference was detected among the homolgous peptides obtained from amylase I and amylase II. This result does not account for the difference of 5 aspartic acid and/or asparagine residues observed at the amylase level. Two hypotheses to interpretate this discrepancy are discussed.

STAPHYLOCOCCAL NUCLEASE AND ITS COMPLEXES WITH NUCLEOTIDIC INHIBITORS - A PHOTO-CIDNP STUDY OF AROMATIC RESIDUES EXPOSURE

A laser photo‐CIDNP study at 360 MHz of Staphylococcal nuclease shows that calcium binding induces a switch in the accessibility of Tyr85 and Tyr115. This switch, which can be reverted by subsequent addition of pdTp inhibitor, might be relevant to the induction of nucleolytic activity upon calcium binding. The conformations of the ternary complexes of calcium (containing nuclease with pdTp, dTpCH2dTp and dTpCH2dTpCH2dT) are very similar and are interpreted in terms of comparable binding patterns at the active site of the enzyme.