Susanne Klumpp

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 K i, 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.

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 (K i app = 0.16 nM) more potently than protein phosphatase 2A (K i app = 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 μM. 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.

Guanylate cyclase in the excitable ciliary membrane of Paramecium

The ciliate P~~urneci~rn has a cell membrane which responds to environmental stimuli with altered conductances and potentials, the ciliary apparatus being regulated by the rate at which calcium enters the cell [ f ,2]. As demonstrated in deciliation experiments, the calcium inward current during excitation is carried through voltage-sensitive ion channels in the surface membrane covering the cilia f3,4]. The behavioral correlate of the Ca2+/K” action potential is the known avoiding reaction of Paramecium. The excitable ciliary membrane resembles that of neurons, the Ca*+/K’ action potential being comparable to the NdfK+ action potential in higher nervous systems. Cilia of Paramecium can be isolated and biochemical processes involved in motility and membrane excitability can be investigated. There is convincing evidence that cyclic nucieotides and Ca’+ serve as interrelated second messengers in a number of cellular systems ]5]. Here we report the presence of a particulate guanylate cyclase (EC 4.6 I 1.2, GC) localized in the excitable ciliary membrane of Paramecium. [ar-32P]-GTP (1 I.tCi, from Amersham) and 50-100 pg protein. Formation of cGMP was determined by liquid scintilIation counting with correction for recovery of c ]“H] GMP (from Amersham) after chromatographic separation of GTP and cGMP on alumina columns as in [9]. Production of cGMP was linear in respect to time and protein. cGMP identity was verified by thin-layer chromatography and radioautography, Protein was estimated by the Lowry method using bovine serum albumin as standard.

Cloning and expression of a bovine adenylyl cyclase type VII specific to the retinal pigment epithelium.

A cDNA of a type 7 adenylyl cyclase isoform was cloned from a bovine retinal pigment epithelium cDNA library using oligonucleotides developed to conserved regions common to mammalian adenylyl cyclases. A 6.7 kb mRNA of very high abundance was uniquely present on Northern blots containing mRNA or total RNA from the pigment epithelium. This transcript was undetectable in all other tissues examined. The cDNA encoded a protein of 1,097 amino acids and exhibited the known doublet of 6 transmembrane‐spanning regions in a hydrophobicity plot. The novel member of the type 7 adenylyl cyclase isoform was expressed in COS‐1 cells. It was stimulated 10‐ and 20‐fold by 10 μM GTPγS and 100 μM forskolin, respectively. The high expression rate exclusively in the retinal pigment epithelium suggests that this adenylyl cyclase isoform is involved in processes specific to this functionally exceedingly important subretinal cell layer.

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.

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.

Effect of tryptic calmodulin fragments on guanylate cyclase activity from paramecium tetraurelia

Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.

Biochemistry of Cilia

In the 1960s and 1970s, biochemical investigations of Paramecium concentrated mainly on isozyme patterns of esterases, phosphatases, succinate-, isocitrate-, glutamate-, and β-hydroxybutyrate dehydrogenases, and fumarase (Tait 1970a, b; Esteve 1970; Allen and Gibson 1971; Allen and Nerad 1978a; Khadem and Gibson 1985). The isozyme patterns were preferably used as taxonomic tools. Earlier, the surface antigens of Paramecium were also a major area of biochemical research efforts (Beale and Kacser 1957; Hansma and Kung 1975; Steers and Davis 1977). These large immobilization proteins (i-antigens) are dealt with by Schmidt in Chapter 11 (this Vol.). Starting around 1970, Paramecium was recognized as a possible model cell for a genetic dissection of behavior (Kung 1971a, b; Kung and Eckert 1972). The advantage of using Paramecium was that both the genetics and electrophysiology of this genus were reasonably well understood at that time (Kung et al. 1975). Biochemistry was added to this model system as a new dimension in 1976 (Browning and Nelson 1976). In 1977, it became clear that the membrane covering the motile machinery of Paramecium, about 5000 cilia, carries the voltage-dependent Ca channels which are responsible for the depolarizing Ca-inward current, and possibly for other ion channels as well (Ogura and Takahashi 1976; Dunlap 1977). Since then, the cilia and in particular their excitable membrane, which comprises about two-thirds of the total surface membrane of Paramecium (Andrews and Nelson 1979), have been the topic of numerous biochemical studies aimed at forming an idea of how ion channel regulation and translation of an ionic signal into a mechanical response, i.e., ciliary reversal, and how desensitization and adaptation are accomplished in molecular terms.