Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling

We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency.     

Kinetic behavior of porcine pancreatic phospholipase A2 on zwitterionic and negatively charged double-chain substrates

A number of isomeric diacylglycerophosphocholines and diacylglycero sulfates containing O-acyl and/or S-acyl ester bonds were investigated as substrates for porcine pancreatic phospholipase A2 and its zymogen. A comparison is made with the kinetic properties of the enzyme toward the corresponding glycol detergents previously described [van Oort, M. G., Dijkman, R., Hille, J. D. R., & de Haas, G. H. (1985) Biochemistry (preceding paper in this issue)]. Hydrolysis of the secondary ester bond in the 1,2-diacylglycero-3-type lipids proceeds much faster than the splitting of the primary ester function present in the isomeric 1,3-diacylglycerol and 1-acylglycol derivatives. In sharp contrast to the glycol detergents, the substitution of the cleavable oxygen ester by a thio ester bond in the glycerol lipids results in 5 times lower kcat values. At alkaline pH and above the critical micelle concentration, the anionic sulfates are much better substrates than the corresponding phosphocholine-containing detergents. At very low detergent concentrations, below the critical micelle concentration, the anionic sulfates induce protein aggregation such that phospholipase A2, as well as its zymogen, is present in high molecular weight complexes containing several protein molecules. In these aggregates, protein-protein and/or lipid-protein interactions strongly activate phospholipase but not the zymogen.     

Studies on the specificity of the cholesterol-binding pancreatic proteinase and identification as human pancreatic elastase 1

The proteolytic attack of the cholesterol-binding pancreatic proteinase (CBPP) on the oxidized insulin A and B chains as well as on glucagon was investigated by kinetic studies. The reaction products were isolated by high-pressure liquid chromatography and identified by amino acid analysis. The combined results reveal a pronounced selectivity of CBPP for the peptide bonds at the carboxy ends of Ala, Val, Leu, Ser, His and Thr residues with Ala, Val and Leu most favoured, indicating a close catalytic relationship to porcine pancreatic elastase [Narayanan, A. S. & Anwar, R. A. (1969) Biochem. J. 114, 11-17] and the anionic porcine pancreatic protease E [Kobayashi R., Kobayashi, Y. & Hirs, C. H. W. (1981) J. Biol. Chem. 256, 2460-2465] which resembles human pancreatic elastase 1. The immunological comparison indeed disclosed the identity of CBPP with human pancreatic elastase 1.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.     

Identification of residues within the extracellular domain 1 of bovine Fc gamma 2R essential for binding bovine IgG2

Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFc gamma 2R initiates the killing process. bFc gamma 2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian Fc gamma Rs, bFc gamma 2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and Fc alpha RI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715-1722) that like these proteins (and unlike other Fc gamma Rs), bFc gamma 2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFc gamma 2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F-G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.     

Biosynthesis of salivary proteins in the parotid gland of the subhuman primate, Macaca fascicularis. Cell-free translation of the mRNA for a proline-rich glycoprotein and partial amino acid sequence and processing of its signal peptide

The major anionic proline-rich proteins in the parotid and submandibular secretions of subhuman primates and man perform the important biological function of inhibiting crystal growth of calcium phosphate salts from saliva, which is supersaturated with calcium phosphate salts, thereby preventing excess deposition of hydroxylapatite on tooth surfaces. The present work was initiated as a first step towards investigating proline-rich protein biosynthesis in parotid glands using the subhuman primate, Macaca fascicularis, as a model system. RNA was isolated from macaque parotid glands and separated into poly(A)-enriched and poly(A)-deficient fractions by chromatography on oligo(dT)-cellulose. The mRNAs in both fractions promoted incorporation of radiolabeled amino acids into polypeptides in an mRNA-dependent reticulocyte lysate translation system. Five major proline-rich polypeptides were detected and one of these was shown to be the in vitro precursor of the major anionic macaque proline-rich protein (MPRP), which is the structural and functional counterpart of the major anionic proline-rich proteins in the parotid and submandibular secretions of man (Oppenheim, F.G., Offner, G.D., and Troxler, R.F. (1982) J. Biol. Chem. 257, 9271-9282). Radiosequencing of the material in anti-MPRP immune precipitates showed that the in vitro precursor of MPRP contained an 18-residue signal peptide. The in vitro precursor of MPRP was processed in dog pancreas vesicles to a form with a lower apparent Mr and with an NH2-terminal amino acid sequence identical to that of native MPRP. The phenylthiohydantoin derivatives of Ala and Ile were detected at residue 9 and those of Val and Met were detected at residue 16 of the signal peptide. This indicated that the in vitro precursor of MPRP, which migrated electrophoretically as a single band in anti-MPRP immune precipitates, contained two different in vitro polypeptides derived from two different mRNAs. These results are discussed in the context of the genetic polymorphism among the major anionic proline-rich proteins in the parotid and submandibular secretions of man.     

Complex of digestive proteinases of <Emphasis Type="Italic">Galleria mellonella</Emphasis> caterpillars. Composition,properties, and limited proteolysis of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> endotoxins

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.     

A low-molecular-mass Kazal-type protease inhibitor isolated from rat hepatocytes is identical to rat pancreatic secretory trypsin inhibitor II. Purification and amino acid sequence

A low-molecular-mass serine protease inhibitor was purified from hepatocytes and liver of rats. It was found to be a single polypeptide of 56 amino acid residues corresponding to Mr = 6224, a value that is in agreement with the molecular mass determined by gel chromatography. The inhibitor formed a complex in a molar ratio of 1:1 with trypsin. Its complete amino acid sequence was identical with that of pancreatic secretory trypsin inhibitor II (PSTI-II) in pancreatic juice, but not with that of PSTI-I [Uda, K., Ogawa, M., Shibata, T., Murata, A., Mori, T., Kikuchi, N., Yoshida, N., Tsunasawa, S. & Sakiyama, F. (1988) Biol. Chem. Hoppe-Seyler 369, 55-61]. PSTIs have been reported to be primarily pancreatic secretory products, but in have been reported to be primarily pancreatic secretory products, but in patients immunoreactive PSTI was found in the plasma and urine during acute inflammatory disease and shown to be produced ectopically in cancer tissues. Here we report for the first time that PSTI-II is present in other normal tissues besides the pancreas.     

The 4F2 antigen heavy chain induces uptake of neutral and dibasic amino acids in Xenopus oocytes.

The 4F2 cell surface antigen is a disulfide-linked heterodimer induced during the process of cellular activation and expressed widely in mammalian tissues (Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931). The human heavy chain component, a type II membrane glycoprotein, has 29% identity to the amino acid transport-related protein encoded by the recently cloned rat D2 cDNA. We have demonstrated that Xenopus oocytes injected with in vitro transcribed cRNA from D2 take up cystine and dibasic and neutral amino acids (Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600). In the present study, we examine the role of the human 4F2 heavy chain in amino acid transport. In vitro transcribed 4F2 cRNA was injected into Xenopus oocytes which were assayed for the uptake of radiolabeled amino acids. Our results show that cRNA from 4F2 stimulates the uptake of dibasic and neutral amino acids into oocytes at levels up to 3-fold higher than for water-injected control oocytes. There is no demonstrable uptake of cystine. Uptake is saturable, with characteristics of high affinity transport, and inhibition data suggest that uptake occurs via a single transporter. Dibasic amino acids are taken up by both 4F2 and D2 cRNA-injected oocytes in a sodium-independent manner. In contrast, 4F2-induced but not D2-induced neutral amino acid uptake has a significant component of sodium dependence. Likewise, neutral amino acids in excess inhibit the 4F2-induced uptake of radiolabeled arginine but not leucine in a sodium-dependent manner. The 4F2-induced uptake we observe most likely represents the activity of a single transport system with some characteristics of systems y+, b0,+, and B0,+. We suggest that 4F2 and D2 represent a new family of proteins which induce amino acid transport with distinct characteristics, possibly functioning as transport activators or regulators.     

Energization of amino acid uptake by system A in cultured human fibroblasts

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.     

Differential interfacial and substrate binding modes of mammalian pancreatic phospholipases A2: a comparison among human, bovine, and porcine enzymes.

To identify the residues essential for interfacial binding and substrate binding of human pancreatic phospholipase A2 (hpPLA2), several ionic residues in the putative interfacial binding surface (R6E, K7E, K10E, and K116E) and substrate binding site (D53K and K56E) were mutated. Interfacial affinity of these mutants was measured using anionic polymerized liposomes, and their enzymatic activity was measured using various substrates including phospholipid monomers, zwitterionic and anionic micelles, and anionic polymerized mixed liposomes. Similar mutations (R6E, K10E, K56E, and K116E) were made to porcine pancreatic phospholipase A2 (ppPLA2), and the properties of mutants were measured by the same methods. Results indicate that hpPLA2 and ppPLA2 have similar interfacial binding mechanisms in which cationic residues in the amino terminus and Lys-116 in the carboxy terminus are involved in binding to anionic lipid surfaces. Small but definite differences between the two enzymes were observed in overall interfacial affinity and activity and the effects of the mutations on interfacial enzyme activity. The interfacial binding of hpPLA2 and ppPLA2 is distinct from that of bovine pancreatic phospholipase A2 in that Lys-56 is involved in the interfacial binding of the latter enzyme. The unique phospholipid headgroup specificity of hpPLA2 derives from the presence of Asp-53 in the substrate binding site. This residue appears to participate in stabilizing electrostatic interactions with the cationic ethanolamine headgroup, hence the phosphatidylethanolamine preference of hpPLA2. Taken together, these studies reveal the similarities and the differences in the mechanisms by which mammalian pancreatic phospholipases A2 interact with lipid aggregates and perform interfacial catalysis.     

Amino acid sequence of a proline-rich phosphoglycoprotein from parotid secretion of the subhuman primate Macaca fascicularis

The complete amino acid sequence of the macaque proline-rich phosphoglycoprotein (MPRP) was determined by automated Edman degradation of the protein, fragments F-1 and F-2 derived from the protein by an intrinsic salivary protease, and chymotryptic, tryptic, Staphylococcus aureus V8 protease, and endoproteinase lysine-C peptides. MPRP contains 115 amino acid residues including phosphorylated serine at residues 1, 2, 6, 12, and 15, and 6 O-glycosidic carbohydrate units at residues 69, 75, 87 (threonine) and 96, 103, and 106 (serine). The Mr of the polypeptide moiety of the protein is 12,656. The amino-terminal domain contains all 5 phosphoserine residues and most of the other negatively charged and hydrophilic residues, whereas the carboxyl-terminal domain contains 24 of 25 proline residues, and 6 O-glycosidic oligosaccharides. Comparison of MPRP with the four major anionic proline-rich proteins (PRPs) from human glandular secretion shows that 57% of the amino acid residues are identical if gaps are introduced to maximize homology, suggesting that these proteins are phylogenetically related. Significant structural and functional differences occur between the macaque and human proteins. MPRP has 5 phosphoserines, PRPs have 2. MPRP is a glycoprotein, PRPs are not. MPRP inhibits the spontaneous precipitation (primary precipitation) of calcium phosphate salts from supersaturated solutions in addition to inhibiting seeded crystal growth (secondary precipitation) (Oppenheim, F. G., Offner, G. D., and Troxler, R. F. (1982) J. Biol. Chem. 257, 9271-9282), whereas PRPs inhibit only secondary precipitation. MPRP is the only major anionic proline-rich protein in macaque glandular secretion; in contrast, there are four major anionic PRPs and these display a genetic polymorphism. The significance of these structural differences with respect to biological function and the possible relationship of MPRP to salivary mucins are discussed.     

Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets

Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.     

The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.     

Respiratory proteins from the extremely thermophilic aerobic bacterium, Thermus thermophilus. Purification procedures for cytochromes c552, c555,549, and c1aa3 and chemical evidence for a single subunit cytochrome aa3

We have developed a chemically defined, minimal growth medium for Thermus thermophilus which is suitable for nutritional studies, isotopic enrichment, and genetic manipulation of the organism. Reliable procedures are described for the large scale purification of cytochrome c552 from the periplasm and for cytochrome c555,549 and cytochrome c1 aa3 from the plasma membrane. In contrast to a previous report (Fee, J. A., Choc, M. G., Findling, K. L., Lorence, R., and Yoshida, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 147-151) which suggested a molecular weight near 200,000, the cytochrome c1aa3 complex was shown by protein and amino acid analyses to have Mr approximately 93,000. Sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography, combined with amino acid analyses, revealed the presence of only two proteins in a 1:1 ratio: C-protein has Mr approximately 33,000, binds heme C, and is thought to correspond to cytochrome c1. A-protein has Mr approximately 55,000 and is thought to bind the four redox components (2 heme A and 2 Cu) of cytochrome aa3.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A2

The amino acid composition and partial NH2-terminal amino acid sequence of the phospholipase A2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH2-terminal 24 amino acids were found to be as follows: X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp- Val-Ser-Tyr-Gly-Phe-Tyr-Gly- The enzymes contains 5Phe, 8Met, 9Ile, 24Tyr, and 25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A2. This is the first report of the tentative characterization of a eukaryotic phospholipase A2, the cellular source of which is known, i.e., it does not originate from a venom or the pancreas.     

Isolation and characterization of rat pancreatic elastase

Proelastase has been purified to homogeneity from rat pancreatic tissue by a combination of CM-Sephadex and immobilized protease inhibitor affinity resins. Trypsin activation yields an elastolytic enzyme that possesses a specificity toward small hydrophobic residues in synthetic amide substrates, similar to those of porcine elastase 1 and canine elastase. However, the rat enzyme also rapidly hydrolyzes a substrate containing tyrosine in the P1 position. N-Terminal sequence analysis reveals that rat proelastase has an identical activation peptide with that of porcine proelastase 1 and has two conservative amino acid sequence differences from the activation peptide of canine proelastase. The sequence data established that rat proelastase corresponds to the elastase 1 mRNA clone isolated by MacDonald et al. [MacDonald, R. J., Swift, G. H., Quinto, C., Swain, W., Pictet, R. L., Nikovits, W., & Rutter, W. J. (1982) Biochemistry 21, 1453]. The sequence and substrate data obtained for rat and canine elastases suggest that there is a family of pancreatic elastases with properties similar to those of the classically described porcine elastase 1.