Okadaic acid (OA) and calyculin A (CLA), which are potent and specific inhibitors of serine/threonine protein phosphatases type 1 and 2A, have been shown to induce drastic changes in platelet morphology. The aim of this study was to analyse the molecular mechanisms of OA- or CLA-induced cytoskeletal reorganization, with a specific focus on microtubules and actin filaments. Confocal fluorescence microscopy revealed that OA or CLA altered the distribution of microtubules from marginal band arrangements to homogeneous patterns, consistent with the transmission-electron-microscopic finding that microtubules were fragmented and redistributed into pseudopod-like processes. In thrombin-activated platelets, OA or CLA induced extremely long pseudopods containing an array of microtubules and actin filaments, and a condensed mass of actin filaments in the centre of platelets. OA or CLA induced the constriction of actin filaments without an increase in filamentous (F)-actin, and also rather significantly inhibited actin polymerization in thrombin-activated platelets. Furthermore, neither OA or CLA enhanced phosphorylation of myosin light chain (MLC). By immunoprecipitation of platelet lysate with anti-alpha-tubulin antibody, a 90 kDa protein was co-precipitated with tubulin and was predominantly phosphorylated in the presence of OA. As the time-dependent phosphorylation of 90 kDa protein correlated well with the reorganization of microtubules, these data suggest that phosphorylation and dephosphorylation of this protein might play a role in the regulation of microtubule organization. These findings indicate that OA or CLA induces reorganization of microtubules and actin filaments via the phosphorylation of a microtubule-associated 90 kDa protein and an MLC-phosphorylation-independent mechanism. mechanism.
Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1. Efficient extraction of this enzyme from the crude material was possible using "Cellulosin AP," a microbial polysaccharidase preparation containing cellulase, hemicellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31,000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS.
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