Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidinecontaining peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 lmolAEmin )1 AEmg )1 at pH 7.5 with 7 lM phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNAlibrary followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 lmolAEmin )1 AEmg )1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.Keywords: dephosphorylation; N-phosphorylation; phosphoamidase; phosphopeptide; protein histidine phosphatase.Boyer and coworkers detected protein-bound phosphohistidine in rat-liver mitochondrial succinyl-CoA synthetase almost 40 years ago [1,2]. Despite the long time interval and the fact that phosphohistidine represents a substantial fraction of eukaryotic protein-bound phosphate [3], only a few phosphohistidine-containing proteins have been detected compared to the large number of eukaryotic proteins phosphorylated on serine, threonine and tyrosine residues. One reason for this difference may be that the N-bound phosphate of phosphohistidine easily escapes detection by common analytical procedures, due to its lability under acidic conditions, e.g. during fixation and staining of gels after SDS/PAGE [4].The studies on eukaryotic protein histidine phosphorylation and dephosphorylation have dealt with essentially two aspects. One is the intermediary phosphorylation of enzymes [5][6][7][8][9][10], of which nucleoside diphosphate kinase is a particularly well-studied example. The other is the reversible protein histidine phosphorylation by protein kinases and phosphatases [3,11]. An important contribution to the latter field was the purification of a yeast protein histidine kinase in 1991 [12]. Access to this enzyme also made possible the preparation of 32 P-labelled histone H4, which was later used as substrate in the search for phosphohistidine phosphatases. Using such an approach, the catalytic subunits of the well-studied serine/...

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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.

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 aminoterminal 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.The discovery of an extralysosomal tripeptide-releasing amino-peptidase in rat liver has been reported (1). Its substrate specificity, as studied by use of a series of peptide substrates, is complex (1, 2). The peptidase releases tripeptides with little apparent similarity, although the rate of cleavage varies considerably. The enzyme, currently named tripeptidyl peptidase II (TPP-II) (2, 3), has a high native molecular weight (>106) (1), a subunit molecular weight of 135,000, and a neutral pH optimum (2). The presence ofTPP-II in numerous tissues of the rat (2) and in erythrocytes and liver of several other species (4) has been demonstrated. The human erythrocyte enzyme has been classified as a serine peptidase due to its complete inhibition by diisopropyl fluorophosphate (iPr2P-F) and phenylmethylsulfonyl fluoride (2). However, the enzyme was also inhibited by some of the thiol reagents tested, e.g., N-ethylmaleimide, p-chloromercuribenzoate, and Hg2+. TPP-II could be protected against iPr2P-F, phenylmethylsulfonyl fluoride, or N-ethylmaleimide by the use of a competitive inhibitor (2), suggesting that not only a serine, but also a cysteine residue is of importance for the activity of the enzyme. To obtain an unambiguous classification of TPP-II as a serine peptidase, we considered it necessary to isolate and determine the amino acid sequence of the part of the protein that contains the active serine residue. Information on the primary structure of the active site would also be ofinterest for the investigation of structural relationships between TPP-II and other peptidases. EXPERIMENTAL PROCEDURES Materials. [1,3-3H]iPr2P-F was purchased from AmershamInternational. Emulsifier scintillator 299 came from Packard. Dithiothreitol and bovine trypsin were from Boehringer Mannheim, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin...

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