Sequential homologies between procarboxypeptidases A and B from porcine pancreas

Automated Edman degradation of monomeric procarboxypeptidases A and B from porcine pancreas shows that their N-terminal regions (from residue 1 to 34-37) present a high degree of sequential homology to each other as well as to other related procarboxypeptidases. Conformational predictions based on these sequences confirm their structural homology and indicate the probable existence of two beta-turns, one beta-chain and a long alpha-helix in them. On the other hand, tryptic peptide maps on a reverse-phase column indicate great sequential similarities (if not identity) between monomeric procarboxypeptidase A and the procarboxypeptidase A subunit isolated from its binary complex with proproteinase E.     

The isolation and partial characterization of precursor forms of ostrich carboxypeptidase

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.     

The average conformation at micromolar [Ca2+] of Ca2+-atpase with bound nucleotide differs from that adopted with the transition state analog ADP.AlFx or with AMPPCP under crystallization conditions at millimolar [Ca2+

Crystalline forms of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP.fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1.AMPPCP and the E1.ADP.AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mM Ca2+ (the concentration prevailing in the crystallization media) than when they are examined in the presence of the few micromolar of Ca2+ known to be sufficient to saturate the transport sites. This concerns various properties, including ATPase susceptibility to proteolytic cleavage by proteinase K, ATPase reactivity toward SH-directed Ellman's reagent, ATPase intrinsic fluorescence properties (here described for the E1.ADP.AlFx complex for the first time), and also the rates of 45Ca2+-40Ca2+ exchange at site "II." These results solve the above paradox at least partially and suggest that the presence of a previously unrecognized Ca2+ ion in the E1.AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1.AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1.AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, toward the transition state leading to phosphorylation and Ca2+ occlusion.     

Existence of ternary complexes of procarboxypeptidase A in the pancreas of some ruminant species

The existence of procarboxypeptidase A, in the form of a non-covalent ternary complex containing the apparently inactive serine protease (subunit III), has so far been observed only in the ox pancreas. Evidence, obtained in the present study, shows that a ternary complex of procarboxypeptidase A, with a subunit III highly homologous with that of the bovine complex, is also present in two other ruminant species, sheep and goat. The biological significance of these complex forms of procarboxypeptidase A and the consistently high biosynthesis level of the apparently inactive subunit III in all three ruminant species is still unknown. Yet the synthesis of subunit III is not related to the animal diet since in the horse, which is a non-ruminant herbivorous animal, the procarboxypeptidase A is monomeric. Reassociation assays between either bovine subunits II or III and monomeric as well as binary forms of procarboxypeptidase A from various species show that, unlike subunit II, the recognition site for subunit III is highly conserved in all the procarboxypeptidases A and that bovine subunit II is different from porcine chymotrypsinogen C with regard to association.     

Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms.

Bovine liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been obtained in three forms designated transferase I, A and B on the basis of their elution positions from chromatography on phosphocellulose. All forms have been shown to have a molecular weight of about 152 000, each being composed of four similar subunits. Amino acid analysis of transferase A and B, the two major forms, revealed a close relationship between both forms with almost identical amino acid composition and arginine as N-terminal residue. The three transferases differ with respect to their redox state and their multiplicity of forms with isoelectric points of 6.9, 7.5 and 8.8, into which the transferases I and A were spontaneously transformed upon isoelectric focusing or rechromatography on phosphocellulose. Transferase B represents a stable enzyme form with an isoelectric point of 8.8. Although the redox state of transferase B can be adjusted to that of transferase A still a difference in charge and in the multiplicity of forms exists, thus indicating different protein states.     

Aspartate aminotransferase isoenzymes in Leptosphaeria michotii. Properties and intracellular location

Two forms of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five-step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 9110 nkat oxalacetate/mg protein-1 and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers of Mr 92,000. They differed mainly in their Km for 2-oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer of Mr 25,000; form B with an unidentified protein of Mr 40,000-44,000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form B [corrected] and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasm. Moreover, mitochondria were isolated and purified by Percoll-density-gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.     

Immunochemical aspects, molecular and kinetic properties of multiple forms of acetyl-CoA acetyltransferase from rat liver mitochondria.

Acetyl-CoA acetyltransferase (EC 2.3.1.9) from rat liver mitochondria, which catalyzes the first step in the biosynthesis of ketone bodies, exists in two forms, designated transferase A and transferase B. Both transferases showed immunochemical cross-reactivity, but are immunologically unrelated to cytosolic acetyl-CoA acetyltransferase activity and the mitochondrial acetyl-CoA acyltransferase from rat liver. The transferases A and B were estimated to have molecular weights of 151 000 in the absence and 40 000 in the presence of sodium dodecyl sulfate. They differ with respect to charge states and multiplicity of forms as indicated by isoelectric focusing. Transferase A appeared in two forms with isoelectric points of 8.4 and 9.1, whereas transferase B represents a stable protein state with an isoelectric point of 9.0. Kinetic analysis of the reactions leading to acetoacetyl-CoA synthesis revealed saturation curves with multiple intermediary plateaus, indicating a complex kinetic behaviour. The data presented are interpreted as representing a microheterogeneity of forms of the mitochondrial acetyl-CoA acetyltransferase. The kinetic properties exhibited suggest a role for this microheterogeneity in the regulation of ketogenesis.     

Resolution by high-pressure liquid chromatography and partial characterization of multiple forms of cytochrome P-450 from hepatic microsomes of phenobarbital-treated rats

Seven cytochromes P-450 (A, B, C, D, E1, E2 and F) were isolated from hepatic microsomes of phenobarbital-induced rats by a modification of the procedure of Guengerich and Martin [Arch. Biochem. Biophys. (1980) 205, 365-379]. The modification consisted of replacing DEAE-cellulose column by two DEAE-Sepharose CL-6B columns connected in tandem, changing the elution scheme and monitoring the resulting fractions by high-pressure liquid chromatography (HPLC). Cytochrome P-450 forms D, E1, E2 and F having molecular masses of 52.5 kDa, 52.5 kDa, 53.3 kDa and 53.2 kDa, respectively were resolved from the major form of cytochrome P-450 'peak B2' of Guengerich and Martin (above reference). These four cytochromes P-450 were immunologically identical by Ouchterlony double-diffusion analysis. Slight but significant differences were evident in the partial peptide digest maps of these four cytochromes P-450 and catalytic properties of these four forms, though qualitatively similar, demonstrated distinct quantitative differences. Furthermore, HPLC retention times of these four cytochrome P-450s were quite different. Cytochrome P-450 forms A, B and C were distinctly different from each other and from the forms D, E1, E2 and F in the following respects: partial peptide digest maps, catalytic activities, and HPLC retention times. The present results show that cytochromes P-450 considered homogeneous by sodium dodecyl sulfate/polyacrylamide gel electrophoresis may be heterogeneous and contain multiple forms of cytochromes P-450 with different net charges but similar molecular-masses. These studies also demonstrate the capability of HPLC in providing a simple and effective tool for monitoring the separation of cytochromes P-450 showing charge heterogeneity.     

Purification of a carboxypeptidase B-like enzyme from the starfish Dermasterias imbricata.

A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters of lysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.     

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).     

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.     

Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography.

Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.     

Analysis of the structure of T4 bacteriophage-modified valyl-tRNA synthetase by limited proteolysis and isoelectric focusing.

The new form of valyl-tRNA synthetase (EC 6.1.1.9) that appears immediately after infection of Escherichia coli with bacteriophage T4 was purified and subjected to mild proteolysis using five different proteases. The inactivation of aminoacylation activity was both more extensive and rapid than that obtained with valyl-tRNA synthetase purified from uninfected E. coli. The addition of bulk tRNA from E. coli B protected the phage-specific form of valyl-tRNA synthetase from proteolysis, but ATP and valine did not exhibit a similar protective effect. The characteristic property of phage-modified valyl-tRNA synthetase, resistance to denaturation by 4 M urea, remained unaffected during treatment with trypsin. This suggested that the phage-specific factor tau, known to be associated with the synthetase in phage-infected cells, was protected from proteolysis in the synthetase-tau complex. Comparison by isoelectric focusing of normal valyl-tRNA synthetase, the phage-specific form of this enzyme, and phage enzyme from which tau had been removed, revealed no differences in the isoelectric points of these three molecules. Based on these results a model was drawn for the structural changes occurring in valyl-tRNA synthetase after association with the phage factor tau.     

Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."     

Comparison of the multiple molecular forms of bovine adrenocortical P450scc with those of corpus luteum P450scc.

1. Bovine adrenocortical P450scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450scc of the same molecular size and the P450scc could be resolved into 3-4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450scc were more complex than those of the corpus luteum P450scc. The corpus luteum P450scc was practically devoid of the neutral to acidic isoelectric point forms. 3. Three to four P450scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. The structural basis of the multiple molecular forms was discussed.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Genetic and biochemical aspects of sucrase from Drosophila melanogaster

Isoelectrofocusing of abdominal extracts of Drosophila melanogaster revealed the existence of two forms of sucrase (E.C. 3.2.1.26). One form exhibited an isoelectric point of 4.63 +/- 0.02 while the other form exhibited an isoelectric point of 4.83 +/- 0.02. The localization of the structural gene for sucrase is proposed on the basis of enzyme determinations in a series of duplication- and deletion-bearing aneuploids. We suggest that the sucrase structural gene lies between 31CD and 31EF on the left arm of chromosome 2 and that the two forms of abdominal sucrase derive from a common protein coded for by a single sucrase gene designated Sucr+.     

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Chymotrypsin C is a co-activator of human pancreatic procarboxypeptidases A1 and A2

Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94-96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as chymotrypsin B1, chymotrypsin B2, chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.     

Equus przewalskii plasma protease inhibitor (Pi) system

A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) were found to control the plasma Pi proteins of the E. przewalskii haplotypes. In contrast a fourth locus, Spi 4, was found in some E. caballus haplotypes. The significance of these results with respect to the complexity of the protein pattern exhibited by the equine Pi multigene family is discussed.