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E Waelkens

Publications and source records attributed to E Waelkens.

54 records · Page 3Linked to original sources
PubMed1992

Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase.

The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49].

Actin Cytoskeleton↗
PubMed1989

Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens.

A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC). On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups. Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone. The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests. The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification.

Acinetobacter↗
PubMed1987

The interrelationship between cAMP-dependent alpha and beta subunit phosphorylation in the regulation of phosphorylase kinase activity. Studies using subunit specific phosphatases.

This study addresses the function of multisite phosphorylation of phosphorylase kinase catalyzed by the cAMP-dependent protein kinase. Using subunit specific protein phosphatases (the polycation-stimulated and ATP-, Mg2+-dependent enzymes), we show that the degree of phosphorylation of both the alpha and beta subunits modulates phosphorylase kinase activity. beta subunit phosphorylation is essential for activation and, independent of the degree of alpha subunit phosphorylation, enzyme fully dephosphorylated in the beta subunit is completely inactivated. alpha Subunit phosphorylation does, however, also regulate activity, and enzyme fully or partially phosphorylated in the beta subunit is inactivated as a consequence of alpha subunit dephosphorylation. The extent of inactivation caused by alpha subunit dephosphorylation is linearly dependent on the phosphorylation state of the beta subunit. Three peptide sites on the alpha subunit are phosphorylated by the cAMP-dependent protein kinase; the site primarily affecting activity is the one that is initially phosphorylated. These data provide evidence that subunit interrelationships play an important role in the regulation of phosphorylase kinase by multisite phosphorylation.

Adenosine Triphosphate↗
PubMed1987

Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle.

Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.

Animals↗
PubMed1987

Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases.

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.

Adenosine Triphosphate↗
PubMed1987

The polycation-stimulated protein phosphatases: regulation and specificity.

Four classes of protein phosphatases are presumed to play an important role in dephosphorylating the major proteins involved in the control of general metabolism. Based on the enzyme-directed regulation of activity they have been classified as ATP,Mg-dependent-, polycation-stimulated-, Mg2+-dependent protein phosphatases and calcineurin. We have recently purified from rabbit skeletal muscle four distinct PCS protein phosphatases, classified according to the apparent molecular weight of the native enzymes in gel filtration at an early stage of the purification as: PCSH (390 kDa), PCSM (250 kDa) and PCSL (200 kDa) phosphatases. The PCSH phosphatase could be resolved into a 3(65:55 35 kDa)-subunit PCSH1 phosphatase and a 2(65:35 kDa)-subunit PCSH2 enzyme probably derived from the PCSH1 phosphatase, both characterized as specific deinhibitor phosphatases. PCSM phosphatase, a 3(72:65 35 kDa)-subunit enzyme, shows a high degree of stimulation with a low concentration optimum of polycations and is sensitive to a Ca2+-dependent protease, which brings about a five- to ten-fold increase in inhibitor-1 phosphatase activity. PCSL phosphatase is characterized by a 2(65:35 kDa)-subunit structure, a low intrinsic deinhibitor phosphatase activity and a low degree of stimulation of phosphorylase phosphatase activity requiring high concentrations of polycations. At low concentrations of polycations the stimulation of phosphorylase phosphatase activity of the PCS enzymes is enzyme-directed, since it occurs at concentrations far below the substrate concentration. The degree of stimulation is also typical for each type of enzyme (PCSM greater than PCSH1 greater than PCSH2 greater than PCSL greater than PCSC) and dependent on the polycation used; at the optimum concentration the most effective polycations (polylysine, protamine, histone H1) stimulate the phosphorylase phosphatase activity to about the same extent. Polycation concentrations above the optimum are less effective on phosphorylase phosphatase activity and can even become inhibitory to the basal activity. Whether this effect is enzyme- or substrate-directed (or both) is not known. The stimulation by polycations could be completely lost following preincubation of the PCS phosphatase with polycations. This deactivation is time-, temperature- and concentration-dependent. However the polycations did not affect the basal phosphorylase phosphatase activity. In addition to phosphorylase a and inhibitor-1, casein, myosin light chains and phosphorylase b kinase (alpha-subunit) are choice substrates for these enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Characterization of the catalytic subunits of the different types of polycation-stimulated protein phosphatases.

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.

Animals↗
PubMed1986

Phosphorylation of the modulator protein of the ATP, Mg-dependent protein phosphatase by casein kinase TS. Reversal by PCS phosphatases and control by distinct phosphorylation site(s).

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.

Adenosine Triphosphate↗
PubMed1986

Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle.

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.

Adenosine Triphosphate↗
PubMed1985

Activation of the PCSM-protein phosphatase by a Ca2+-dependent protease.

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.

Animals↗
PubMed1985

Dephosphorylation of the deinhibitor protein by the PCSH 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.

Adenosine Triphosphate↗
PubMed1984

Inhibitor-1 phosphatase activity in vascular smooth muscle.

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.

Animals↗
PubMed1984

The deinhibitor protein: regulation by phosphorylation-dephosphorylation.

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.

Animals↗
PubMed1984

Regulation of protein phosphatase activity by the deinhibitor protein.

In liver and muscle the major active phosphorylase and synthase phosphatase activity is associated with the glycogen particle. When we examined the effect of the inhibitor-1 and modulator protein on the enzyme present in crude glycogen fractions from dog liver, the phosphorylase phosphatase was not or only slightly affected. Since the enzyme isolated from the glycogen complex by DEAE-cellulose chromatography could be inhibited by inhibitor-1 as well as the modulator protein, it was assumed that an unknown mechanism or factor present in the glycogen fraction was responsible for this reduced sensitivity of the protein phosphatase. This led to the discovery (7) of the deinhibitor protein which has now been extensively purified from dog liver. The deinhibitor protein was shown to be thermostable, ethanol- and trichloroacetic acid-resistant, but non-dialyzable and it was destroyed by pronase or trypsin. The apparent molecular weight was estimated at about 17,500 in gel filtration, 8,300 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and 5,500 in sucrose density gradient centrifugation, behavior which is consistent with the assumption that the deinhibitor protein may have little ordered structure. Glycogen synthesis requires both phosphorylase and glycogen synthase as dephosphorylated enzymes. The interaction of the deinhibitor protein with the protein phosphatase brings about several effects which, when considered together, could all facilitate the dephosphorylation of glycogen synthase and phosphorylase. The protein phosphatase present in a resuspended glycogen pellet dephosphorylates inhibitor-1 in the absence of Mn2+. This ability of the phosphatase, which is lost during purification of the enzyme, can be restored upon addition of the deinhibitor protein. Owing to the association of the deinhibitor protein with the active phosphatase the enzyme becomes insensitive to inhibition by inhibitor-1 and the modulator protein, and more resistant to the conversion into the FA-ATP,Mg-dependent form, brought about by the modulator protein. During the activation of the ATP,Mg-dependent phosphatase under conditions where kinase FA is rate limiting, the deinhibitor protein increases the level without affecting the rate of activation.

Adenosine Triphosphate↗
PubMed1983

Modulation of latent protein phosphatase activity from vascular smooth muscle by histone-H1 and polylysine.

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.

Animals↗
PubMed1983

Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase.

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.

Adenosine Triphosphate↗
PubMedDate not supplied

Photodynamic treatment of basal cell carcinoma and squamous cell carcinoma with hypericin.

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.

Adult↗