Use of a dehydroalanine-containing peptide as an efficient inhibitor of tripeptidyl peptidase II.

Tripeptidyl peptidase II is an intracellular exopeptidase, which has been purified from rat liver and human erythrocytes. An efficient specific inhibitor was obtained through beta-elimination of phosphate from the phosphopeptide Arg-Ala-Ser(P)-Val-Ala. The dehydroalanine-containing peptide formed was a competitive inhibitor with a Ki of 0.02 +/- 0.01 microM. This study demonstrated that replacing a serine residue in a good inhibitor with a dehydroalanine residue reduced the Ki 45 times. It is proposed that dehydroalanine-containing peptides could be of interest in the development of inhibitors for other peptidases as well.

Phosphoprotein patterns in Onchocerca volvulus developmental stages.

Living females and microfilariae of Onchocerca volvulus incubated in culture medium containing [32P]orthophosphate were observed to phosphorylate their proteins rapidly. Patterns of phosphoproteins in extracts from these labelled parasites were compared after two dimensional electrophoresis and autoradiography. Protein extracts from eggs, microfilariae and adult females of O. volvulus were phosphorylated in the presence of [gamma-32P]ATP, magnesium acetate, and added cyclic AMP-dependent protein kinase or the endogenous protein kinase present in the extracts. Patterns of phosphoproteins were compared after separation by single and two-dimensional gel electrophoresis followed by autoradiography. Common phosphopeptide bands were observed when phosphorylated extracts from adult females, microfilariae and eggs were compared. However, extracts from eggs displayed unique phosphorylated polypeptides of M(r) 30,000 and 34,000 that were absent from the extracts from microfilariae. Furthermore, two phosphorylated polypeptides of M(r) 47,000 and 76,000 were detected in extracts from microfilariae but not from eggs. These results indicate that O. volvulus parasites may phosphorylate different proteins at different stages of their development.

A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase.

The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1' position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1' position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.

Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin.

Tripeptidyl peptidase II (TPP II) is a large intracellular exopeptidase with an active site of the subtilisin type. Affinity-purified hen antibodies against human erythrocyte TPP II cross-reacted with fibronectin in an immunoblot analysis. Furthermore, antibodies against human fibronectin cross-reacted with TPP II. Antibodies against a 65 kDa cell-binding fragment of fibronectin specifically reacted with TPP II, whereas antibodies against the collagen-binding domain, the main heparin-binding domain or the N-terminal fibrin-binding domain did not react. Moreover, the affinity-purified antibodies against TPP II reacted with a 105 kDa cell-binding fragment of fibronectin but not with the fibrin-binding domain or the collagen-binding domain. When native TPP II was dissociated into smaller units through dialysis against a dilute Tris buffer, it could be digested by chymotrypsin into three stable fragments of 70 kDa, 42 kDa and 20 kDa. It could be demonstrated that the 42 kDa fragment was specifically recognized by antibodies against the 65 kDa cell-binding fragment of fibronectin. Furthermore, labelling with di-[3H]isopropyl phosphorofluoridate and N-terminal sequence determination showed that the 70 kDa fragment contained the active-site serine residue. In conclusion, our findings suggest that one domain of the TPP II molecule bears structural resemblance to a cell-binding fragment of fibronectin.

Supramolecular structure of tripeptidyl peptidase II from human erythrocytes as studied by electron microscopy, and its correlation to enzyme activity.

Tripeptidyl peptidase II is an extralysosomal serine peptidase of an unusually large size, i.e. Mr greater than 10(6) for the native enzyme and Mr 135000 for the subunit. The enzyme from human erythrocytes was studied by electron microscopy on samples negatively stained by ammonium molybdate. Two different structural representations of the purified enzyme were obtained, both with a length of about 50 nm, and consisting of repetitive substructures. Upon dialysis of the enzyme against a Tris/HCl buffer, the activity was gradually decreased. This decrease was shown to parallel the dissociation of the large enzyme structures into smaller ones, the smallest measuring 3 nm by 10 nm and apparently corresponding to the repetitive substructures. The results indicate that a large polymeric form of the enzyme is a prerequisite for full activity.

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.

Detection of protein kinase substrates in extracts of Onchocerca volvulus.

Extracts of Onchocerca volvulus were phosphorylated in the presence of (gamma 32P)ATP and Mg2+ by endogenous protein kinase activity and exogenous rabbit muscle catalytic sub-unit of the adenosine 3'5' monophosphate dependent protein kinase (E.C. 2.7.1.37). Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of the 32P-labelled extracts revealed at least seven (32P)-phosphoproteins with apparent Mr of 92,000; 86,000; 40,000; 27,000; 26,000; 23,000 and 17,000. The phosphorylation of the components with apparent Mr of 23,000 and 17,000 was catalysed by both endogenous and exogenous protein kinases, whereas the other components required exogenous protein kinase for their phosphorylation. The endogenous protein kinase activity was inhibited by suramin and the heat-stable protein inhibitor of the adenosine 3'5' monophosphate dependent protein kinase. The (32P)phosphoproteins identified in this investigation are probably candidate regulatory molecules in O. volvulus; though their physiological functions remain to be determined.

Purification, substrate specificity, and classification of tripeptidyl peptidase II.

An extralysosomal tripeptide-releasing aminopeptidase was recently discovered in rat liver (Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. (1983) J. Biol. Chem. 258, 11622-11628). In the present work this tripeptidyl peptidase is shown to occur in several rat tissues and in human erythrocytes. The erythrocyte enzyme was purified about 80,000-fold from a hemolysate while the rat liver enzyme was purified about 4,000-fold from a homogenate. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions more than 90% of the protein was represented by a polypeptide of Mr 135,000 in both cases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed similar specific activity, and had a pH optimum around 7.5. A tryptic pentadecapeptide from the alpha-chain of human hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, was found to be sequentially cleaved by the erythrocyte enzyme into five tripeptides, beginning from the NH2 terminus. Chromogenic tripeptidylamides showed various rates of hydrolysis at pH 7.5. With Ala-Ala-Phe-4-methyl-7-coumarylamide, Km was 16 microM and Vmax 13 mumol min-1 . mg-1, comparable to the standard substrate Arg-Arg-Ala-Ser(32P)-Val-Ala values (Km 13 microM and Vmax 24 mumol . min-1 . mg-1). The tripeptidyl peptidase of human erythrocytes was classified as a serine peptidase from its irreversible inhibition by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala (Ki 1.5 microM). [3H]Diisopropylphosphate was incorporated to the extent of 0.7-0.9 mol/mol of Mr 135,000 subunit, which confirms the high purity of the enzyme.

The role of the phosphate group for the structure of phosphopeptide products of adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

By c.d. studies it is shown that liver-pyruvate-kinase-related peptide substrates of cyclic AMP-dependent protein kinase have a high tendency towards non-random structures in non-aqueous media. When phosphorylated, the conformation tendencies decrease. This structural change is explained in terms of the formation of strong intrapeptide phosphate-guanidinium salt links. It is proposed that similar events occur at the catalytic site of protein kinase and that such an interaction could facilitate the removal of the phosphorylated products.

Tripeptidyl aminopeptidase in the extralysosomal fraction of rat liver.

An enzyme which removes tripeptides from the free, NH2-terminal end of oligopeptides has been detected in the extralysosomal fraction of rat liver. The enzyme was partially purified by Sepharose CL-4B and DEAE-cellulose chromatography. The pH optimum was in the neutral range and the apparent native molecular weight was above 10(6), as judged by the Sepharose chromatography. The enzyme cleaved the phosphopeptide Gly-Val-Leu-Arg-Arg-Ala-Ser(P)-Val-Ala (I), first at the Leu-Arg bond and then at the Ala-Ser(P) bond. The cleavage of the former bond was inhibited by Arg-Arg-Ala-Ser(P)-Val-Ala (II), which indicated that both bonds were cleaved by the same enzyme. Km for II was 0.01 mM at pH 6.5-7.5. Val-Leu-Arg-Arg-Ala-Ser(P)-Val-Ala (III) and Leu - Arg - Arg - Ala - Ser(P)-Val-Ala were poor substrates. III was, however, found to be an efficient inhibitor. The Ala-Ser(P) bond of Arg-Arg-Ala-Ser(P)-Val (IV) was cleaved at the same rate as that of II. The enzyme was active also with the unphosphorylated peptides corresponding to II and IV and tolerated the substitution of lysine for the NH2-terminal arginine of the latter peptide. Substitution of guanidovaleric acid for the NH2-terminal arginine of IV and of guanidovaleric acid or epsilon-amino-hexanoic acid for the NH2-terminal arginine of unphosphorylated IV reduced the rate of hydrolysis to insignificant levels, demonstrating the importance of a free NH2 terminus. The results provide evidence of a unique tripeptidyl aminopeptidase.

Phosphopeptide substrates of a phosphoprotein phosphatase from rat liver.

The substrate specificity of a preparation of phosphoprotein phosphatase (Mr = 32 000) from rat liver was investigated. Phosphopeptides based on the structure Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Glx-Leu and Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys were used. These phosphopeptides correspond to the phosphorylation sites of rat liver pyruvate kinase (type L) and the beta subunit of rabbit muscle phosphorylase b kinase, respectively. A decrease in the apparent Km values and a concomitant increase in Vmax values was observed when the number of amino acyl residues after the phosphoseryl residue in the respective phosphopeptides were increased from 2 to 4, 5, or 6. Most of the phosphopeptides investigated generally showed apparent Km values higher than the values obtained with phosphopyruvate kinase. Ala-Ser(P)-Val-Ala and Gly-Ser(P)-Val-Tyr appeared to be the shortest phosphopeptides that could be dephosphorylated rapidly. These findings support the hypothesis that a small part of the phosphoprotein may be sufficient to fulfill the minimal requirements for its dephosphorylation.