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The pars intercerebralis-corpora cardiaca system (PI-CC) of insects is the endocrinological equivalent of the hypothalamus-pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI-TOF and electrospray ionization double quadrupole time of flight (ESI-Qq-TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI-TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP(3)R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GXGXXG (amino acids 1773-1780 and 2016-2021, respectively). In IP(3)R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920-1925), which is conserved in the three IP(3)R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP(3)-induced Ca(2+) release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.

Identification of substances able to elicit physiological or behavioural processes that are related to reproduction would greatly contribute to the domestication of commercially important crustaceans that do not reproduce easily in captivity. Crustaceans are thought to release urine signals used for chemical communication involved in courtship behaviour. In contrast to insects, very little is known about the endocrinological processes underlying this phenomenon. Therefore, an extract of 3500 central nervous systems of female white shrimp Penaeus vannamei was screened for myotropic activity in order to purify pyrokinin-like peptides that belong to the pyrokinin/PBAN neuropeptide family. Members of this family regulate reproductive processes in insects, including pheromone biosynthesis. Purification of these pyrokinins was achieved by a combination of reversed-phase and normal-phase chromatography. Subsequent characterization by mass spectrometry, Edman degradation and peptide synthesis resulted in the elucidation of two novel peptides. Pev-PK 1 has the primary sequence DFAFSPRL-NH(2) and a second peptide (Pev-PK 2) is characterized as the nonapeptide ADFAFNPRL-NH(2). Pev-PK 1 contains the typical FXPRL-NH(2) (X = G, S, T or V) C-terminal sequence that characterizes members of the versatile pyrokinin/PBAN family. Pev-PK 2 displays an Asn residue at the variable X position of the core pyrokinin sequence. These crustacean pyrokinins are the first to be found in a noninsect. The synthetic peptides display myotropic activity on the Leucophaea maderae as well as on the Astacus leptodactylus hindgut.

Plasmodial fragmin, a Physarum polycephalum F-actin severing and capping protein, is phosphorylated by casein kinase II at Ser(266) (De Corte, V., Gettemans, J., De Ville, Y., Waelkens, E., and Vandekerckchove, J. (1996), Biochemistry 35, 5472-5480). In this study, we report the purification and characterization of the corresponding fragmin phosphatases. One of the enzymes was purified to near homogeneity from a cytosolic extract; it dephosphorylates CKII-phosphorylated fragmin, a peptide encompassing the CKII phosphorylation site of fragmin as well as histone 2A, CKII-phosphorylated casein and the CKII model-peptide substrate: R(3)E(3)S(P)E(3). Its activity was highly stimulated by Mn(2+) and Mg(2+), and based on its lack of sensitivity toward phosphatase effectors we could exclude similarities with PP1, PP2A and PP2B phosphatases. All biochemical properties of the phosphatase point to a PP2C-like enzyme. A second phosphatase dephosphorylating fragmin was identified as a Physarum alkaline phosphatase., (Copyright 2000 Academic Press.)

Purpose: We determined the sensitivity and specificity of detecting flat bladder carcinoma in situ through fluorescent detection after intravesical hypericin instillations., Materials and Methods: The study included 40 patients, of whom 26 presented with macroscopic visible tumor, 9 had a positive cytology without visible tumor and 5 underwent cystoscopy after bacillus Calmette-Guerin instillations (4) or radiotherapy (1). We instilled 40 ml. of a 8 microM. solution of hypericin intravesically for at least 2 hours. Fluorescence excitation with blue light was effective up to 16 hours after termination of the instillation., Results: All visible papillary tumors showed red fluorescence. In addition, 134 flat fluorescent areas were detected. Analysis of 281 biopsies from flat bladder wall indicated 93% sensitivity and 98.5% specificity for detecting carcinoma in situ. Visible lesions resulting from radiotherapy, chemotherapy or immunotherapy did not show any fluorescent signs and, therefore, did not induce false-positive readings. There were no signs of photobleaching during inspection and resection., Conclusions: We report a simple yet comprehensive endoscopic method for early detection of bladder cancer, including carcinoma in situ. Hypericin induced fluorescence has a high sensitivity and specificity for detection of bladder transitional cell carcinoma, papillary and flat carcinoma in situ. When carcinoma in situ is suspected, this technique is highly recommended.

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.

The neuropeptides inducing dark color in albino nymphs of the migratory locust Locusta migratoria were isolated from the larval brain of the silkworm, Bombyx mori and from the adult corpora cardiaca (CC) of the cricket Gryllus bimaculatus, respectively, and their amino acid sequences identified. The two peptides isolated from the two different species are identical to [Arg(7)] corazonin, a neuropeptide known to be present in a cockroach and others. This peptide induces a dark color in albino nymphs of L. migratoria at fmol levels, and a high dose of >/=100 pmol caused albino locusts to turn completely black, but it influenced neither body color nor metamorphosis in B. mori and G. bimaculatus. Therefore, the physiological functions of [Arg(7)] corazonin in the silkworm and the cricket remain unknown. The present study demonstrated the usefulness of the albino strain of L. mirgatoria as a specific bioassay system for this peptide.

Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.

Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

This paper reports the purification of a tachykinin isoform from the midgut of the desert locust, Schistocerca gregaria. One hundred locust midguts were extracted in an acidified methanolic solvent, after which three HPLC column systems were used to obtain a pure peptide. A tachykinin immunoassay was used to monitor all collected fractions. After each purification step the purity of the sample was monitored by MALDI-TOF mass spectrometry. The pure peptide was sequenced by ESI-Qq-oa-TOF mass spectrometry. Edman degradation-based automated microsequencing and chemical synthesis confirmed the sequences. The midgut peptide, GNTKKAVPGFYGTRamide (Scg-midgut-TK), belongs to the tachykinin family with identified members in all vertebrate phyla and some invertebrate phyla: arthropods, annelids and molluscs. Scg-midgut-TK is the first tachykinin purified from midguts of the desert locust Schistocerca gregaria. In comparison to locust brain tachykinins, the midgut tachykinin is N-terminally extended. Similar to neuropeptide gamma, an N-terminally extended mammalian tachykinin, first isolated from rabbit intestine, the present identified locust intestinal tachykinin contains a putative dibasic cleavage site., (Copyright 1999 Academic Press.)

Recently, we have partially purified and characterized a specific cell cycle-regulated cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step (R. Derua, I. Stevens, E. Waelkens, A. Fernandez, N. Lamb, W. Merlevede, and J. Goris, 1997, Exp. Cell Res. 230, 310-324). In the present paper we describe its purification to homogeneity. We could identify the kinase as a special form of calcium/calmodulin-dependent protein kinase II (CaMKII), consisting of five isoforms with molecular masses ranging from 52 to 83 kDa. At least three of them could be considered as novel. Using an in vivo assay with a synthetic peptide (cyktide), an activation of the kinase was shown at about 50% maturation. Further evidence for this observation came from the injection of the calcium chelator BAPTA and the specific cyk/CaMKII inhibitor AIP. A delay of oocyte maturation of at least 1 h was observed. Besides serine 53, a second cyk phosphorylation site in cyclin B2 was identified as threonine 41. Site-directed mutagenesis of these sites indicated that phosphorylation of these sites in Xenopus cyclin B2 was not required for the hallmark functions of cyclin B2., (Copyright 1999 Academic Press.)

In the third step of the alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant.

In response to crowding, locusts develop characteristic black patterns that are well discernible in the gregarious phase at outbreaks. We report here a dark-color-inducing neuropeptide (dark-pigmentotropin) from the corpora cardiaca of two plague locusts, Schistocerca gregaria and Locusta migratoria. The chromatographic isolation of this neuropeptide was monitored by using a bioassay with an albino mutant of L. migratoria. The neurohormone, consisting of 11 amino acids, is identical to [His7] corazonin, previously isolated from corpora cardiaca of another acridid without known function. The present results show that even in isolated (solitary) nymphs, [His7] corazonin induces gregarious black patterns. Its primary structure shows some similarity with the vertebrate melanophore stimulating hormone.

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.

Kinins comprise a family of peptides that were first found in the central nervous system of insects and recently also in mollusks and crustaceans. After the isolation of the first members of the kinin family, the leukokinins from Leucophaea maderae, leukokinin-related peptides were found in the cricket Acheta domesticus and the locust Locusta migratoria, all through their ability to induce Leucophaea maderae hindgut contraction. Subsequently, kinins were found in the mosquitoes Culex salinarius and Aedes aegypti and in the earworm Helicoverpa zea. The first noninsect member of this family was isolated from a mollusk, the pond snail Lymnaea stagnalis. Most recently our group has isolated the first kinins from crustaceans. Six kinins were isolated from the white shrimp Penaeus vannamei. To date, 35 members of this family have been isolated. The first relatively small family of insect kinins has grown into an expanding and rather large family with members in insects, crustaceans, and mollusks. In this paper we discuss the kinin family in terms of method of isolation, structure, in vitro and in vivo activity, distribution, receptors, and signal transduction. We will compare the crustacean and insect members of the kinin family, using the data available on crustacea.

Changes in actin cytoskeleton in the C6 rat glial cell line were studied during decrease or increase (abrupt or gradual) of extracellular osmolality. Actin cytoskeleton was visualized by confocal microscopy after FITC-phalloidin labeling. G-actin, Triton-soluble F-actin and Triton-insoluble F-actin subfractions were determined by gel electrophoresis and scanning, and by DNase I inhibition assays. In control conditions C6 glial cells exhibited well-defined stress fibers and a relatively smooth cortical network. Extracellular anisosmotic changes induced a rapid actin cytoskeletal reorganization, which further progressed and was not reversed upon cell volume recovery. Hypotonic shock caused membrane ruffling and a shift towards polymerized actin, whereas hypertonicity (abrupt or gradual) led to a distinct morphological appearance of abundant short actin microfilaments with, however, no detectable alteration in actin subfractions. When anisosmotic cell volume regulation was prevented, cytoskeleton reorganization depended on the osmotic change and the experimental protocol, but was not related to the absence of volume readjustment. Therefore, although involvement of cytoskeletal alterations in transduction of volume regulatory responses cannot be excluded, it is likely that the observed changes in actin cytoskeleton in C6 glial cells are linked with, but do not initiate, cell volume regulatory processes.

Hypericin displays antiproliferative and cytotoxic effects on tumor cells. This effect depends on photodynamic activation with visible light and oxygen. Hence, we explored its potential use in treating skin cancer. Eight patients with squamous cell carcinoma (SCC) and eleven patients with basal cell carcinoma (BCC) were treated topically with hypericin. After intralesional injection, the hypericin was irradiated with visible light. Patients with SCC were given 40-100 micrograms hypericin intralesionally, 3-5 times per week for 2-4 weeks; patients with BCC 40-200 micrograms hypericin 3-5 times per week for 2-6 weeks. Hypericin displayed selective tumor-targeting: penetration in the surrounding tissues did not induce necrosis or cell loss and even the generation of a new epithelium at the surface of the malignancy was noticed. The effectiveness of the therapy depends on the concentration and total dose of hypericin, the frequency and duration of the therapy; clinical remissions can be expected after 6-8 weeks.

This paper reports the purification of three myotropic neuropeptides from the white shrimp Penaeus vannamei. The central nervous systems of 3500 shrimps were extracted in an acidified solvent, after which four to five HPLC column systems were used to obtain pure peptides. A cockroach hindgut muscle contraction bioassay was used to monitor all collected fractions. The pure peptides were submitted to Edman degradation based automated microsequencing. Mass spectrometry and chemical synthesis confirmed the sequences. Ala-Pro-Ser-Gly-Phe-Leu-Gly-Met-Arg-NH2 (Pev-tachykinin, 934.1 Da) belongs to the tachykinin family with identified members in all vertebrate classes and some invertebrate classes: arthropods, annelids and molluscs. A very specific Pev-tachykinin antiserum was developed, which labels 4 neurosecretory cells in the brain. Ala-Ser-Phe-Ser-Pro-Trp-Gly-NH2 (Pev-kinin 1, 749.8 Da) and Asp-Phe-Ser-Ala-Trp-Ala-NH2 (Pev-kinin 2, 694.7 Da) are the first crustacean kinins. Pev-kinin 2 is the first kinin with a Trp-Ala-NH2 instead of a kinin-typical Trp-Gly-NH2 carboxyterminus., (Copyright 1998 Academic Press.)

A protease-inhibitor was isolated from mature ovaries of Schistocerca gregaria by a combination of trypsin-affinity chromatography and reverse-phase high performance liquid chromatography. It was characterized by aminoterminal amino acid sequencing using Edman degradation based automated microsequencing and by MALDI-TOF mass spectrometry. The N-terminal sequence (Y)XAEXDELA(A)EEY(Y)Q(Q)X(I)(L)M (X being a Cys, an irregular or modified amino acid) revealed no similarities with any other protease inhibitors isolated from invertebrate or vertebrate source. The 14 kDa inhibitor was found to be heat-stable. It shows potent inhibitory activity toward bovine trypsin and chymotrypsin, but not toward pancreatic elastase. It is likely that the characterized inhibitor will serve as an important tool for understanding its role in insect development., (Copyright 1997 Academic Press.)

We have partially purified a specific cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step. Phosphopeptide analysis identified Ser53 as the major in vitro phosphorylation site for cyk in cyclin B2. Using a synthetic peptide derived from cyclin B2 encompassing Ser53 (cyktide) as a substrate, cyk was shown to be activated during progesterone-induced maturation, with a peak of activity between 40 and 50% maturation. A sustained high cyk activity was observed in oscillating egg extracts. Microinjection of cyk-phosphorylated cyclin B2 into prophase oocytes accelerated progesterone-induced maturation by about 2 h, indicating that cyclin B2 is a relevant substrate for cyk and that the function of cyk is situated upstream of cdc2-cyclin B activation. Microinjection of cyk-phosphorylated cyktide or a combination of cyk and cyclin B1 into G2 fibroblasts induced significant changes in cell morphology, reminiscent of a premature prophase-like phenotype. Similarly, addition of cyk-phosphorylated cyktide in cyclin B1-dependent interphase extracts resulted in histone H1 kinase activation.

Actin-fragmin is a heterodimeric protein complex from Physarum polycephalum microplasmodia that is phosphorylated in vitro at residues Thr203 and Thr202 of the actin subunit by the endogenous actin-fragmin kinase. Following phosphorylation, the F-actin capping activity of the complex becomes Ca(2+)-dependent, suggesting a fundamental regulatory role in controlling F-actin growth [Gettemans, J., De Ville, Y., Waelkens E. and Vandekerckhove, J. (1995) J. Biol. Chem. 270, 2644-2651]. In this study we analysed actin phosphorylation in vivo. We demonstrate that the actin-fragmin complex constitutes the only substrate of the actin-fragmin kinase in plasmodia. Monomeric actin is not phosphorylated. Immunoprecipitation of actin-fragmin reveals that approximately 40% of the actin subunit of the complex is phosphorylated in vivo. However, using purified substrate and kinase, the complex can be quantitatively phosphorylated as judged by two-dimensional gel electrophoresis. Through comparative phosphopeptide fingerprinting, we show that the phosphorylation sites in vivo are identical to those identified in vitro. We additionally characterized a complex of actin and the NH2-terminal half of fragmin (residues 1-168) that is also phosphorylated by the same kinase. In contrast to actin-fragmin, phosphorylation of the complex between actin and residues 1-168 of fragmin is independent of Ca2+ because the second Ca(2+)-dependent regulatory actin-binding domain is missing. By artificially varying the actin-fragmin concentration or the actin-fragmin kinase activity present in microplasmodia cytosolic extracts, we attempted to detect alternative protein substrates for the actin-fragmin kinase. The fact that none could be identified suggests that the control and properties of actin-fragmin phosphorylation observed in vitro may stand as a model for F-actin growth control in Physarum cells.

Fragmin is a 42 kDa regulatory protein involved in actin microfilament organization in Physarum polycephalum. We show that fragmin is a target of casein kinase II (CK II) enzymes isolated from evolutionarily divergent species. In Physarum microplasmodia, two such kinases were identified. A serine residue located in the sequence Gly-Gly-Ser-Asp-Leu-Glu constitutes the phosphorylation site and was identified by phosphopeptide sequencing, mass spectometry analysis, and inhibition studies with a synthetic peptide corresponding to this site. Interestingly, the actin-fragmin dimer (A--F) as well as the actin2-fragmin trimer (A2--F) are equally efficient targets, and phosphorylation had no effect on the actin-binding properties of fragmin. Actin-fragmin isolated from microplasmodia revealed a minor acidic fragmin isoform, suggesting that fragmin is phosphorylated in vivo. The actin-fragmin complex is also phosphorylated on the actin subunit by an endogenous actin-fragmin kinase [Gettemans, J., De Ville, Y., Vandekerckhove, J., & Waelkens, E. (1992) EMBO J. 11, 3185-3191]. We show that the two phosphorylation events act independently of each other.

The 80-kDa actin-fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500-fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polyclonal antibodies raised against four tryptic peptides of the purified 80-kDa AFK recognized the 80-kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78-kDa protein, also displaying AFK activity, appeared to be derived from the 80-kDa AFK. A partial amino acid sequence analysis, covering up to 20% of the protein (150 amino acids), was performed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK.

The inhibitor-1 phosphatase but not the phosphorylase phosphatase activity of a newly discovered 250 kDa polycation-stimulated (PCSM) protein phosphatase in rabbit skeletal muscle is increased up to 10-fold by a Ca2+-dependent protease. The enzyme-directed protease effect to which the PCSH and PCSL phosphatases are insensitive was progressively lost during purification of the enzyme. This could be explained by either a slow conversion of the enzyme to an active form of the enzyme with a change in specificity, or the loss of a protease-sensitive inhibitor of the inhibitor-1 phosphatase activity, resulting in a PCS phosphatase characterized by its high inhibitor-1/phosphorylase alpha activity ratio. The Ca2+-dependent protease is completely inhibited by EGTA or leupeptin.

An apparently latent phosphatase which migrated as a protein of Mr 130,000 during sucrose density centrifugation, and a spontaneously active phosphatase (Mr 68,000) were isolated from bovine aortic smooth muscle. Basal phosphorylase phosphatase activity of the latent preparations was stimulated 12 fold by low concentrations of lysine-rich histone-H1 (30 micrograms/ml) and 6 fold by polylysine (Mr 17,000; 12 micrograms/ml), whereas the spontaneously active enzyme was only slightly affected. The enzymatic activity of the spontaneously active preparation was completely destroyed by beta-mercaptoethanol. In contrast, the apparently latent enzyme was converted to a more active form of lower molecular weight (Mr 86,000) following treatment with beta-mercaptoethanol and this form of the enzyme was still stimulateable by histone-H1. These findings show that the aortic spontaneous and apparently latent phosphatase actives are ascribable to separate enzymes and they suggest that the activity of latent phosphatase in living cells may be modulated by cationic proteins such as histones or similar effector molecules.

The deinhibitor protein, which protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein, stabilizes the enzyme in its active conformation preventing its conversion to the ATP,Mg-dependent enzyme form and controls the dephosphorylation of inhibitor-1, was shown to exist under active and inactive forms. It can be inactivated by the catalytic unit of the cyclic AMP-dependent protein kinase and reactivated by an inhibitor-1 phosphatase, also described as histone-H1 ("latent") stimulated protein phosphatase.

The deinhibitor protein, responsible for the decreased sensitivity of the ATP,Mg-dependent protein phosphatase to inhibitor-1 and the modulator protein, is inactivated by cyclic AMP-dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP,Mg-dependent phosphatase in its activated or spontaneously active form, four different forms of polycation-stimulated phosphatases (PCSH, PCSM, PCSL and PCSC) and calcineurin. Only the high -Mr polycation-stimulated protein phosphatase (PCSH), but not its catalytic subunit (PCSC), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCSH is affected neither by polycations nor by Mn ions.

The phosphorylation by casein kinase TS (II) of the modulator protein of the ATP, Mg-dependent phosphatase increases after preincubation with the PCSH1 phosphatase or with the catalytic subunit of the ATP, Mg-dependent phosphatase. Dephosphorylation by the two phosphatases combined leads to the incorporation of 2 mol phosphate per mol modulator (at Ser residues). Occupancy of the ATP, Mg-dependent phosphatase phosphorylation site(s) is a negative determinant in the phosphorylation of the modulator by kinase TS. Among the PCS phosphatases PCSH1 shows the highest activity toward the 32P-Ser residues labeled by kinase TS in untreated or previously dephosphorylated modulator, while the ATP, Mg-dependent phosphatase is totally ineffective. Protamine stimulates all phosphatase activities, so that the catalytic subunit of the ATP, Mg-dependent phosphatase becomes almost as effective as the PCSC phosphatase in dephosphorylating the kinase TS sites.

Three polycation-stimulated (PCSH-, PCSM- and PCSL-) protein phosphatases are characterized by distinct specificities and regulatory properties. The properties of the catalytic subunits obtained from the 3 basic types of PCS phosphatases are apparently identical. The 35 kDa catalytic subunits are insensitive to inhibitor-1 and modulator protein and in contrast with the holoenzymes are less sensitive to stimulation by protamine, displaying a similar degree of stimulation and an identical concentration optimum; preincubation with polycations also results in a time-dependent deactivation. The phosphorylase phosphatase activity of the three catalytic subunits is stimulated to a similar extent by low but comparable concentrations of detergents, but not by metal ions. Upon limited proteolysis by trypsin the basal, but to a lesser extent the polycation-stimulated activity of the holoenzymes and the catalytic subunits is decreased.

The histone-H1 and polylysine stimulated "latent" phosphorylase phosphatase, characterized by a molecular weight of 260,000 in gel filtration and 130,000 in sucrose density gradient centrifugation has been identified as a major inhibitor-1 phosphatase in vascular smooth muscle. Its substrate: protein phosphatase inhibitor-1, was also shown to be present in the same tissue. Following treatment with beta-mercaptoethanol the enzyme dissociates into a lower molecular weight (38,000) form with a higher specific activity.

The small molecular weight (+/- 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase FA. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.