Hisataka Kasai

Stimulation of cyclic nucleotide phosphodiesterases from rat brain by activator protein, proteolytic enzymes and a vitamin E derivative

Abstract DEAE-cellulose column chromatography of the 105 000 × g supernatant from rat brain homogenate showed the presence of three forms of cyclic AMP phosphodiesterase (3′:5′-cyclic-AMP 5′-nucleotidohydrolase, EC 3.1.4.17) : P1, P2 and P3 enzymes according to their elution order from the column. Cyclic GMP phosphodiesterase activity was found only in P2. The P2 enzyme was stimulated 2–6-fold over the basal activity by three different types of agents; Ca 2+ -dependent activator protein, sodium α-tocopheryl phosphate (a vitamin E derivative) and proteolytic enzymes such as trypsin and chymotrypsin. Unlike the activator protein, the last two types of activators exerted their effects regardless of the presence of Ca 2+ . In contrast to P2, P3 was unaffected by any of the above-mentioned activators, whereas the activity of the P1 was slightly enhanced in the presence of each activator. Upon activating P2 with various activators, a decrease of the K m value for substrate (cyclic AMP or cyclic GMP) and an increase of maximum velocity were commonly observed. Treatment of the brain supernatant with trypsin caused a substantial decrease in the molecular weight of activator-sensitive cyclic AMP phosphodiesterase; the molecular weight of the native (activator-dependent) enzyme was estimated to be approximately 150 000 by Sephadex G-200 gel filtration, while the trypsin-treated enzyme had a molecular weight of 80 000 and was insensitive to activator protein. These activator-dependent and activator-independent enzymes were separated by an affinity column, which was prepared by cyanogen bromide-activated Sepharose with purified activator protein.

Protein analyses and reagents: microscale assay of calcium-binding activity of proteins and peptides using a nitrocellulose membrane

A simple and reliable method for the measurement of calcium binding to proteins and peptides was developed. It is composed of two procedures--filtration through a nitrocellulose membrane filter and estimation of 45Ca retained on the membrane. The routine assay was completed within a few minutes, and only microgram amounts of samples were necessary. This method permitted the quantitative determination of the calcium-binding activity of proteins and peptides including one with a molecular weight of as low as 4000. This method also permitted the detection of low-affinity interactions (Kd congruent to 10(-3) M), possibly because the nitrocellulose membrane did not show nonspecific binding of calcium and because a washing step was not employed in the routine assay. Ultrafiltration membranes when used in the apparatus gave no useful data.

An analytical and preparative method for peptide separation by high-performance liquid chromatography on a macroreticular anion-exchange resin.

Abstract This communication presents a high-performance liquid chromatography system which can be used for analytical peptide mapping as well as for preparative peptide separations. The method uses a macroreticular anion-exchange resin with an organic solvent elution system, and the eluates are monitored directly by uv absorption. Complex peptide mixtures derived from partial hydrolysis of proteins are effectively resolved within 60 min with high reproducibility, and the eluted peptides can be analyzed for amino acid composition without any desalting processes.

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin.

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).