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S Klumpp

Publications and source records attributed to S Klumpp.

At least 37 records · Page 2Linked to original sources
PubMed1996

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.

Amino Acid Sequence↗
PubMed1994

A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. Purification, characterization, and cloning.

We isolated the first membrane-bound type 2C serine/threonine protein phosphatase from the ciliated protozoan Paramecium tetraurelia (PtPP2C). Three isozymes of 33, 32, and 31 kDa with a specific activity of 1 mumol.min-1.mg1 were purified from the ciliary membrane. All enzymatic properties including (a) insensitivity toward inhibitors of other protein phosphatase families such as okadaic acid and microcystin, (b) absolute requirement for divalent cations, and (c) substrate specificity tested with synthetic phosphopeptides were identical to mammalian PP2C enzymes and identified the PtPP2C as a canonical PP2C in spite of it being about 25% smaller. The NH2-terminal was blocked. Microsequencing of six tryptic peptides established a relationship to other PP2C enzymes. The PtPP2C gene was obtained using degenerate oligonucleotide primers and the polymerase chain reaction. The gene coded for a 33-kDa protein with 300 amino acids and had an (A+T) content of 62%, typical for this protozoan. Nine of 15 Gln residues are encoded by TAA, a universal stop codon which codes for Gln in Paramecium. A large truncation at the COOH-terminal is responsible for the smaller size of the PtPP2C. Only a single transcript of 1 kilobase was detected with a Northern blot indicating that the 32- and 31-kDa proteins were proteolytic products of the 33-kDa enzyme. Sequence comparisons with PP2C enzymes from rat, rabbit, yeast, Arabidopsis, and Leishmania defined a highly diverged enzyme family which shares three conserved domains, I, II, and III, accounting for about 25% of the primary structure. We demonstrated further that the distances between domains I/II and II/III are very similar in all PP2C enzymes (9-13 and 74-80 amino acids, respectively). However, the amino acid sequences of the spacer regions are unrelated. In addition, the COOH-terminal ends of 100-200 amino acids which comprise 30-50% of the enzyme, display no identity. A dendrogramm shows that PtPP2C surprisingly is most closely related to the mammalian PP2C, and enzymes from Leishmania, Arabidopsis, and yeast are more distant relatives.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain.

Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach. One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids). The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT. Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids). When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid. As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs. This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis. mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver. It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase.

3T3 Cells↗
PubMed1993

Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant.

Simian immunodeficiency virus (SIV) induces an immunodeficiency syndrome similar to human AIDS. Although the disease course of SIV-induced immunodeficiency is generally measured in months to years, a disease syndrome that results in death in 5 to 14 days has been described in pig-tailed macaques infected with the SIVsmmPBj (PBj) strain. The purpose of this study was to derive an acutely lethal PBj molecular clone in order to study viral genes involved in pathogenesis. Six infectious molecular clones were generated; acutely fatal disease was induced by experimental inoculation of pig-tailed macaques with virus stocks derived from either of two clones, PBj6.6 or PBj14.6. Molecular chimeras were constructed by exchange of regions of the genome of PBj6.6 and a nonlethal, related clone, SIVsmH4. Only a chimera expressing the PBj genome under the control of a SIVsmH4 long terminal repeat induced death soon after inoculation. These studies suggest that multiple viral genes of PBj are critical for development of acute disease. More specifically, the env gene but not the long terminal repeat PBj was required for acute disease induction; however env must act in concert with another gene(s) of the PBj genome.

Animals↗
PubMed1992

Purification, characterization and structure of protein phosphatase 1 from the cilia of Paramecium tetraurelia.

A type 1 serine/threonine protein phosphatase (PP1) which is mostly localized in the excitable ciliary membranes from the protozoan Paramecium, was purified to homogeneity. Approximately 4 micrograms enzyme of 37 kDa was isolated from 100 l axenic culture. The enzymic properties were characterized using phosphorylase a from rabbit skeletal muscle as a substrate and several known effectors of mammalian PP1. The protozoan PP1 was enzymically indistinguishable from its mammalian congener. The amino acid sequence of the Paramecium PP1 was deduced from its cDNA. The full-length clone was obtained in several steps starting with a pair of degenerate primers made according to the two most conserved peptides of rabbit PP1 and PP2A. The gene encodes a protein of 36,392 Da. The identity of the cloned gene and the isolated ciliary PP1 was unequivocally established by microsequencing of four tryptic and cyanogen-bromide peptides which were generated from the purified protein. Paramecium PP1 shows 75% amino-acid-sequence identity with rabbit PP1 alpha. Areas of major differences are the C-termini and N-termini and a sequence between residues 219-242.

Amino Acid Sequence↗
PubMed1992

Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance.

Hyperpolarization of the cell membrane of Paramecium stimulates adenosine 3',5'-monophosphate (cAMP) formation. Manipulations of the K+ resting conductance of the ciliate by adaptation in different buffers affected excitability of the cAMP generating system. Blockade of K+ channels inhibited hyperpolarization-stimulated cAMP formation. A mutant of Paramecium that is unable to control its K+ resting conductance had a defect in cAMP formation. Purified adenylyl cyclase, when incorporated into an artificial lipid bilayer membrane, revealed properties of a voltage-independent K+ channel. This indicates that the adenylyl cyclase of Paramecium has a secondary function as carrier of the K+ resting conductance. A hyperpolarization-activated K+ efflux appears to directly regulate adenylyl cyclase activity in vivo.

Adenylyl Cyclases↗
PubMed1991

Identification of a 42 kDa protein as a substrate of protein phosphatase 1 in cilia from Paramecium.

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.

Animals↗
PubMed1990

Tautomycin from the bacterium Streptomyces verticillatus. Another potent and specific inhibitor of protein phosphatases 1 and 2A.

Tautomycin inhibited the catalytic subunits of protein phosphatase-1 (Kiapp = 0.16 nM) more potently than protein phosphatase 2A (Kiapp = 0.4 nM), and the native forms of these enzymes in mammalian, protozoan and plant extracts were inhibited in a similar manner. Protein phosphatase 2B was inhibited 10,000-fold less potently, while two other phosphatases and six protein kinases were unaffected at 10 microM. Okadaic acid prevented the binding of tautomycin to protein phosphatase 2A, indicating a common binding site for both inhibitors. The different relative potencies of tautomycin and okadaic acid for protein phosphatases 1 and 2A suggest that parallel use of both inhibitors may help to identify physiological substrates for each enzyme.

Antifungal Agents↗
PubMed1990

Chromatographic separation of four Ser/Thr-protein phosphatases from solubilized ciliary membranes of Paramecium tetraurelia by heparin-sepharose.

Chromatography of deoxycholate-solubilized proteins from Paramecium ciliary membranes on heparin-Sepharose resolved three peaks of protein phosphatase activities: one type 2A-like and a type 2C phosphatase in the flow-through fractions, another type 2A-like enzyme in the 0.1 M NaCl eluate and type 1 protein phosphatase in the 0.5 M NaCl eluate. The differential sensitivity of the two type 2A-like phosphatases to heparin and protamine further substantiated the existence of distinct isozymes. Once solubilized, none of these ciliary phosphatases required detergent to remain soluble. The molecular mass as determined by chromatography on Superose 6 was in the range 30,000-45,000 dalton for all four protein phosphatases.

Animals↗
PubMed1990

Echocardiographic detection of an intact aneurysm in a horse.

Two-dimensional real-time echocardiographic examination of a 3-year-old Thoroughbred gelding with pleuropneumonia revealed an intact aneurysm of the right sinus of Valsalva, which was confirmed at postmortem examination. The horse had no clinical signs associated with the aneurysm.

Animals↗
PubMed1990

Guanylate cyclase in olfactory cilia from rat and pig.

A guanylate cyclase was identified in cilia from rat and pig olfactory epithelia. Enzyme activities were 200-250 and 90-100 pmol/min.mg-1, respectively. Activity required the presence of non-ionic detergents, e.g., 0.1% Lubrol PX. MnGTP, not MgGTP was used as a substrate. Furthermore, 0.9 mM free Mn2+ was necessary for optimal activity indicating a regulatory site for a divalent cation. The guanylate cyclase displayed sigmoidal Michaelis-Menten kinetics suggesting cooperativity between MnGTP and enzyme. S0.5 was 160 microM MnGTP. The Hill coefficient of 1.7 indicates that more than one class of substrate-binding sites interact in a positive cooperative manner. ATP inhibited the enzyme and linearized plots of substrate kinetics with MnGTP. SH-Blocking agents reversibly inhibited enzyme activity. Sodium azide and nitroprusside were without effect as were several odorants. A guanylate cyclase activity in cilia from tracheal tissue had properties similar to the olfactory enzyme.

Adenosine Triphosphate↗
PubMed1990

Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants.

The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with Ki values below 0.1 nM. Protein phosphatase 2B is inhibited 1000-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.

Animals↗
PubMed1990

Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization.

A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 microM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 mumols.min-1.mg-1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 microM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 microM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F(-)-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.

Alkaline Phosphatase↗
PubMed1990

Okadaic acid, an inhibitor of protein phosphatase 1 in Paramecium, causes sustained Ca2(+)-dependent backward swimming in response to depolarizing stimuli.

Backward swimming is a stereotypic behavioural response of Paramecium. It is triggered by depolarizing stimuli, which open calcium channels in the excitable ciliary membrane. The influx of Ca2+ causes the reversal of ciliary beat and initiates backward swimming. Here, we demonstrate that the protein phosphatase inhibitor okadaic acid does not affect the normal forward swimming pattern of Paramecium, but greatly extends the duration of backward swimming as initiated by depolarization caused by a rise in extracellular K+. Chelation of external Ca2+ results in an immediate resumption of forward swimming. The results suggest that the voltage-operated calcium channel is inactivated by a dephosphorylation event, and that okadaic acid blocks this dephosphorylation without any effect on the motile apparatus of the cilia. In addition, Paramecium is unique among eukaryotic cells, in that okadaic acid inhibits just one protein phosphatase, namely a type 1 enzyme, 75% of which is tightly associated with the excitable ciliary membrane. The type 2A protein phosphatases in Paramecium are unaffected by okadaic acid. The results indicate that protein phosphatase 1 is the enzyme responsible for the dephosphorylation and closure of the calcium channel in Paramecium.

Animals↗