Resonance assignments and secondary structure of a phytocystatin from Sesamum indicum.

A cDNA encoding a cysteine protease inhibitor, cystatin was cloned from sesame (Sesamum indicum L.) seed. This clone was constructed into an expression vector and expressed in E. coli and purified to homogeneous. The recombinant sesame cystatin (SiCYS) showed effectively inhibitory activity toward C1 cysteine proteases. In order to unravel its inhibitory action from structural point of view, multidimensional heteronuclear NMR techniques were used to characterize the structure of SiCYS. The full (1)H, (15)N, and (13)C resonances of SiCYS were assigned. The secondary structure of SiCYS was identified by using the assigned chemical shifts of (1)H(α), (13)C(α), (13)C(ß), and (13)CO through the consensus chemical shift index (CSI). The results of CSI analysis of SiCYS suggest eight ß-strands (residues 33-46, 51-61, 63-75, 80-87, 150-155, 157-169, 172-183, and 192-195) and two α-helices (residues 16-30, and 120-135).

Endogenous basic protein phosphatases in the brain myelin.

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.

The type-1 protein phosphatase activating factor FA is a membrane-associated protein kinase in brain, liver, heart and muscles.

Although the activating factor FA of the type-1 protein phosphatase has long been recognized as a cytosolic enzyme involved in the regulation of cell metabolism and nervous functions, strong indications have been obtained that FA is in fact a membrane-bound protein kinase in most mammalian tissues. For instance, direct treatment of the tissue extracts including brain, liver, cardiac, smooth and skeletal muscles with 1% Triton X-100 can cause several fold stimulation of the FA activity. Moreover, at least 50% of the FA can be detected in the particulate fractions of the extracts. Chromatography of the extracts in the presence and absence of Triton X-100 further demonstrate these results. The data can now explain the reason why most people can not isolate reasonable amount of FA from mammalian tissues. It is recommended that Triton X-100 should be used for purification of FA from most mammalian tissue extracts. The results also suggest that most previous studies on the action of FA involved in the regulation of cell functions should be re-evaluated and the membrane-associated FA should be taken into consideration.