ABSTRACT
We have investigated the inhibitory effect of K-crown (18-crown-6 potassium picrate) on photosystem II (PSII)-enriched membrane fragments and O2-evolving core complexes. K-crown at 2-4 microM inhibits about half the control level of O2-evolution activity in both types of PSII samples. Oxygen-evolution studies demonstrated that the ether works by inactivating the centres and not by interfering with antenna function or energy transfer to the reaction centre. K-crown does not disrupt binding of the extrinsic proteins associated with O2 evolution nor complex with bound Ca2+ or Cl- cofactors, but rather it directly inhibits electron transfer after the tetrameric Mn cluster. Fluorescence studies on active and Tris-treated samples showed that K-crown does not prevent artificial donors from transferring electrons to PSII but like DCMU inhibits on the acceptor side after QA, the primary quinone acceptor. However, the ether is a leaky inhibitor and may also act as a weak donor when the Mn cluster is not present. Oxygen-production experiments using silicomolybdate as an artificial acceptor (which accepts from both pheophytin and QB in PSII membranes) demonstrated that the inhibition is at or near the DCMU site.
Subject(s)
Binding Sites , Chlorophyll/metabolism , Electron Transport/drug effects , Ethers, Cyclic/pharmacology , Ethyldimethylaminopropyl Carbodiimide/pharmacology , Kinetics , Light , Light-Harvesting Protein Complexes , Molybdenum/metabolism , Oxygen/metabolism , Photosynthesis/drug effects , Photosynthetic Reaction Center Complex Proteins/chemistry , Photosystem II Protein Complex , Plant Proteins/metabolism , Silicon Compounds/metabolism , Spinacia oleracea/metabolismABSTRACT
The major spontaneously active serine/threonine (Ser/Thr) protein phosphatase activities in N. crassa wild type (FGSC 424) were type 1 (PP1), type-2A (PP2A) and type-2C (PP2C). PP1 and PP2C predominantly dephosphorylated phosphorylase a and casein, respectively. PP2A acted on both substrates, but was two-fold more active against casein. PPI activity was inhibited by protamine, heparin, okadaic acid (IC50 50 nM) and mammalian inhibitor- 1 (lC50 2 nM). On the other hand, PP2A activity was inhibited by much lower concentrations of okadaic acid (IC50 0.2 nM) and also by protamine, but not by heparin or inhibitor-l. About 80 per cent of total PP1 activity was associated with the particulate fraction and could be partially extracted with 0.5 M NaCl. Seventy and ninety percent of PP2A and PP2C activities, respectively, were found in the soluble fraction. In addition we have partially purified an acid and thermostable PP1 inhibitor which effectively inhibits both N. crassa and mammalian PP1.
Subject(s)
Animals , Rats , Ethers, Cyclic/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Neurospora crassa/enzymology , Serine/metabolism , Threonine/metabolism , Chromatography , Phosphoprotein Phosphatases/metabolism , Substrate SpecificityABSTRACT
Effect of 1,25,50 and 75 nM of okadaic acid (OA) on human lymphocytes from healthy individuals in culture has been investigated. To our surprise, we observed induction of significantly high sister chromatid exchanges (SCEs) at concentrations known to inhibit both protein phosphatase-1 (PP-1) and protein phosphatase -2A (PP-2A). However, 1 nM okadaic acid, known to inhibit PP-2A alone, did not induce this cellular feature/phenotype. This novel preliminary observation lays foundation for investigating further the role of PP-1 inhibition in governing as yet unknown finer controls in the induction of high SCEs, the mechanism for which has eluded answers till date.
Subject(s)
Cells, Cultured , Enzyme Inhibitors/pharmacology , Ethers, Cyclic/pharmacology , Humans , Lymphocytes/drug effects , Okadaic Acid , Phosphoprotein Phosphatases/antagonists & inhibitors , Protein Phosphatase 1 , Protein Phosphatase 2 , Reference Values , Sister Chromatid Exchange/drug effectsABSTRACT
Stimulation of lamb tracheal smooth muscle fibre strips with endothelin-1 and okadaic acid results in the development of isometric tensions which are long lasting. However, endothelin-1 is more potent constrictor than okadaic acid. An analysis of 20,000 Da regulatory myosin light chain by two-dimensional gel electrophoresis shows an identical phosphorylation pattern.