Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.     

Purification and characterization of pepsinogens and pepsins from Asiatic black bear, and amino acid sequence determination of the NH2-terminal 60 residues of the major pepsinogen

Five pepsinogens were purified to homogeneity from the gastric mucosa of Asiatic black bear and termed pepsinogens I-1, I-2, II-1, II-2, and III. Pepsinogen II-1 was the major component and accounted for more than half of the total pepsinogens. Their molecular weights were estimated to be 40,000 for pepsinogens I-1 and I-2, 38,000 for pepsinogens II-1 and II-2, and 42,000 for pepsinogen III. They resembled each other in amino acid composition, except that pepsinogens I-1 and I-2 contained larger numbers of basic residues than the others. Pepsinogen III was a glycoprotein containing about 3.7% carbohydrate. Each was activated to the corresponding pepsin and their enzymatic characteristics were investigated. The optimal pH against hemoglobin was about 2.2 for pepsin I-1, and about 2.5 for pepsins II-1, II-2, and III. Each pepsin was inhibited by pepstatin as well as porcine pepsin and also by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy)-propane, and p-bromophenacyl bromide. Each pepsin could hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, but the specific activity was much lower than that of porcine pepsin. Activation peptides corresponding to residues 1-43, 1-25, and 26-43 were isolated from an activation mixture of pepsinogen II-1. The amino acid sequences of these peptides and of the NH2-terminal portions of pepsinogen II-1 and pepsin II-1 were determined, resulting in the complete NH2-terminal 60-residue sequence of pepsinogen II-1.     

Monkey pepsinogens and pepsins. VII. Analysis of the activation process and determination of the NH2-terminal 60-residue sequence of Japanese monkey progastricsin, and molecular evolution of pepsinogens

Japanese monkey progastricsin was shown to be activated to gastricsin exclusively by a two-step process through an intermediate form. The occurrence of this process was substantiated by the isolation of the intermediate form and released peptides. By NH2-terminal sequence analyses of these protein and peptide species, the amino acid sequence of the 43-residue activation segment (propart) was determined to be as follows: (Formula: see text) The NH2-terminal 26-residue peptide was released first, resulting in generation of the intermediate form. The subsequent release of peptides, residues Nos. 27-40 and 27-43, generated two gastricsins as the final products. This two-step process of activation of Japanese monkey progastricsin is in striking contrast to the one-step activation process occurring exclusively for pepsinogen A of the same monkey species. The course of molecular evolution of pepsinogens including progastricsins was deduced from the amino acid sequences of their activation segments by constructing phylogenic trees. The trees divided pepsinogens into 3 clusters, i.e., pepsinogens A, progastricsins and prochymosin, showing that these three groups diverged from one another very early on in the course of the evolution of pepsinogens.     

Kinetic studies of the urokinase catalysed conversion of NH2-terminal lysine plasminogen to plasmin.

A method for determining initial velocities of the urokinase (EC 3.4.99.26) catalysed converstion of NH2-terminal lysine plasminogen to plasmin (EC 3.4.21.7) is presented. This reaction has been coupled with the hydrolysis of alpha-N-benzyoly-L-arginine ethyl ester, which is catalysed by plasmin, and its rate has been determined from the time course of the overall reaction. The proenzyme-enzyme conversion was found to obey the Michaelis-Menten rate equation. The following values of the kinetic parameters were obtained: the apparent Michaelis constant, Km = 40.7 +/- 6.2 muM; the catalytic constant, kc = 2.59+/-0.31 s(-1), and kc/Km = 6.36-10(4) +/- 0.24-10(4) M(-1)-s(-1).     

Kinetic studies of the urokinase-catalysed conversion of NH2-terminal glutamic acid plasminogen to plasmin.

Initial velocities for the urokinase (EC 3.4.99.26)-catalysed conversion of glutamic acid plasminogen to plasmin (EC 3.4.21.7) have been determined at various urokinase and glutamic acid plasminogen concentrations. As has been found for the corresponding reaction with lysine plasminogen this conversion obeys the Michaelis rate equation. The apparent Michaelis constants are of the same order of magnitude for lysine and glutamic acid plasminogens. The difference in conversion rates for the reactions has been shown to be connected with their having different catalytic constants. The data were analysed according to two reaction schemes, in one of which only one peptide bond is split during the glutamic acid plasminogen-plasmin conversion and in the other of which the cleavage of two peptide bonds with the obligatory formation of an intermediate plasminogen is assumed. The results favour the former.     

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.     

Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta

Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.     

A comparative study on the NH2-terminal amino acid sequences and some other properties of six isozymic forms of human pepsinogens and pepsins

Six pepsinogen isozymogens, including five forms of pepsinogen A (PGA) and an apparently single form of pepsinogen C (PGC), were isolated simultaneously from the purified total pepsinogen fraction of human gastric mucosa by fast protein liquid chromatography on a Mono Q column, and their NH2-terminal amino acid sequences and some other properties were compared. Upon activation at pH 2.0, all the isozymogens were converted to the corresponding pepsins in a stepwise manner through intermediate forms. The activation rates and the cleavage sites in the activation peptide segment to generate intermediate forms were significantly different among the isozymogens. The NH2-terminal 85-residue amino acid sequences of these isozymogens were determined, including the sequences of the activation peptide segments and the NH2-terminal regions of the corresponding pepsins. Differences in amino acid sequence were found at positions 43 and 77 among the pepsinogen A isozymogens; the residue at position 43 was Lys in PGA-5, PGA-4, and PGA-3a, and Glu in PGA-3 and PGA-2, and the residue at position 77 was Leu in PGA-5 and PGA-4 and Val in PGA-3 and PGA-2. Phosphate was not found in any of the isozymogens. The corresponding pepsins also showed significant variations in properties such as specific activities toward synthetic and protein substrates, pH dependence of activity, susceptibility to various inhibitors, and thermal and alkaline stabilities.     

Appendix. Purification, molecular weight, and NH2-terminal sequence of cystathionine gamma-synthase of Escherichia coli

The cystathionine gamma-synthase of Escherichia coli has been purified to homogeneity. It is a tetramer (Mr = 160,000) composed of identical subunits (Mr approximately 40,000). We have determined its amino acid terminal sequence and thus localized the starting codon of the metB structural gene.     

Human placental sphingomyelinase. Purification to homogeneity, antigenic properties and partial amino-acid sequences of the enzyme

Sphingomyelinase from human placenta was purified to homogeneity in five steps: concanavalin A Sepharose, butyl agarose. Blue Sepharose, sphingosylphosphocholine Sepharose chromatography and FPLC-Mono Q. This lysosomal enzyme has a pH optimum around pH 5.0-6.0. It is a glycoprotein with an approximate molecular mass of 70 kDa which is reduced to 60 kDa by enzymatic deglycosylation. Monospecific antibodies against sphingomyelinase were isolated using sphingomyelinase covalently linked to Sepharose as affinity matrix. These antibodies effectively inhibit the sphingomyelinase activity. Peptides were released from sphingomyelinase by cyanogen bromide or proteolytically by trypsin, proteinase V8 and Lys C for gas phase sequencing. Amino-acid sequences are reported which proved to be the prerequisite for antibody and oligonucleotide screening of the respective human placenta cDNA libraries for the determination of the complete amino acid sequence of human lysosomal sphingomyelinase. In situ hybridisation with a labelled antisense RNA synthesized in vitro using cloned sphingomyelinase-specific cDNA as template, which encodes the peptide sequences described here, revealed the strong expression of sphingomyelinase in human placental villi and normal fibroblasts. Fibroblasts of a Niemann-Pick patient, however, were free of mRNA expressing the sphingomyelinase described here.     

Isolation of human, swine, and rat prepepsinogens and calf preprochymosin, and determination of the primary structures of their NH2-terminal signal sequences

The total RNAs were extracted from human, swine, rat, and calf gastric mucosae, and translated in vitro in the presence of radiolabeled amino acids using a wheat germ cell-free system. Upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the translation products, a protein band with a molecular weight of about 43,000 was obtained in each case as one of the major products. These products could be specifically immunoprecipitated with a corresponding anti-pepsinogen or anti-chymosin antiserum. Radiosequence analysis of these translation products purified by SDS-polyacrylamide gel electrophoresis showed that each of them is a precursor form, i.e., prepepsinogen or preprochymosin, having an amino-terminal extension peptide (signal sequence) comprising 15 (human and swine) or 16 (rat and calf) amino acid residues. The primary structures of these signal sequences were determined to be as follows: (Sequence: see text). These signal sequences share common characteristics with those of other pre-secretory proteins, i.e., the presence of positive charges in the NH2-terminal region, hydrophobic amino acid clusters in the interior part, and amino acids with short side chains at the site of cleavage by the signal peptidase.     

Immunological studies on pancreatic phospholipase A2. Antigenic characterization of the NH2-terminal region.

Rabbit antisera elicited against pure pig, horse, ox, and sheep pancreatic phospholipase A2 revealed considerable immunological differences when tested by double immunodiffusion and microcomplement fixation assays. Snake venom phospholipases did not show any detectable cross-reactions with the pancreatic enzymes. Microcomplement fixation also clearly demonstrated conformational differences between porcine phospholipase A2 and its zymogen. NH2 terminally modified analogs of porcine phospholipase A2 could be clearly distinguished using the same assay. Moreover, strong evidence was obtained that Ala1-Arg6 is a part of an antigenic determinant. Radioimmune assay, using monovalent phospholipase-specific Fab fragments revealed a maximum number of three antigenic sites of phospholipase that can simultaneously be occupied by antibody. The Fab fragments were separated into three fractions, using three immunoadsorbent columns in series. These Fab fractions showed different inhibitory properties toward micellar binding of phospholipase A2. They also exhibited different protective effects against active center modification.     

NH2-terminal analysis of Wolfgram and Folch-Lees proteolipid proteins

Abstract— The NH₂-terminal amino acids of Wolfgram and Folch-Lees proteolipids of bovine and human CNS myelin were determined using the cyanate method (Starke & Smyth , 1963) followed by direct amino acid analysis of the products. Glycine predominated in every case and was recovered in amounts similar to the results described by Whikehart & Lees (1973), who used a dansylation technique followed by thin layer chromatography of the DNS-amino acids. In the present study substantial amounts of glutamic acid, serine, alanine and aspartic acid were also recovered, plus traces of other amino acids. Few differences were observed between Wolfgram and Folch-Lees proteolipids. The end group products of purified W1 proteolipid of bovine Wolfgram fraction, of diazometholysed Folch-Lees proteolipid, and of a sample of phosphatidyl serine had essentially the same composition. The similarity of these results, especially for both fractionated and unfractionated Wolfgram proteolipid, may be evidence that the observed products are derived from phosphoglycerides present in proteolipid rather than from the actual NH₂-terminals of the protein.     

Purification, characterization, and amino acid sequences of pepsinogens and pepsins from the esophageal mucosa of bullfrog (Rana catesbeiana)

Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 32,000-34,000. The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.