Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Energy transfer between fluorescent dyes attached to Ca2+,Mg2+-ATPase in the sarcoplasmic reticulum

Sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was solubilized with a nonionic detergent, dodecyl octaethyleneglycol monoether (C12E8), at a weight ratio of detergent to protein of greater than 10, so that the Ca2+, Mg2+ dependent ATPase existed mainly in a monomeric form (7). The solubilized ATPase was reacted with 10 microM N-1-P or 5 microM DACM in the presence of 5 mM CaCl2, 0.4 M KCl, 20% glycerol and 50 mM TES at pH 7.5 and 20 degrees C. Under these conditions, about 1 mol of N-1-P was incorporated into 10(5) g SR protein on 10 min incubation and 1 mol of DACM was incorporated into the same amount of SR on 5 min incubation. Analysis of the tryptic digest of the N-1-P- or DACM-labeled. ATPase on SDS polyacrylamide gel revealed that almost all the fluorescence was associated with the 30K m.w. subfragment of the ATPase protein. Even when the amount of the probe incorporated into SR-ATPase was increased from 1 to 3 mol per 10(5) g SR protein, all was incorporated into the 30K subfragment. Both the activities of formation and decomposition of the phosphorylated intermediate (EP) were unaffected by these modifications. When the separately labeled ATPases were mixed together in the presence of C12E8 and the detergent was removed by incubation with Bio-Beads SM-2, a significant amount of fluorescence energy transfer was observed between N-1-P and DACM. However, energy transfer did not occur when the labeled ATPases were mixed after removal of C12E8.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of leukotriene D4 catalyzed by lysosomal cathepsin H of rat liver

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.     

Suppressive effect of elcatonin, an eel calcitonin analogue, on excessive urinary hydroxyproline excretion in polyostotic fibrous dysplasia (McCune-Albright's syndrome)

A 12-year-old Japanese girl with polyostotic fibrous dysplasia and endocrine concomitants, was treated with elcatonin, a synthetic eel calcitonin analogue, 10 MRC unit/twice a week given by intramuscular injection. Significant decreases in 24 hr urinary content of hydroxyproline and other amino acids from bone collagen were observed during the course of treatment over 5 months. This biochemical result suggests that the synthetic eel calcitonin analogue exhibits the therapeutic effect in patients with polyostotic fibrous dysplasia by inhibiting bone resorption.     

Effect of high osmotic media on blood viscosity and red blood cell deformability

Effects of high osmotic media on the shape and deformability of RBC were examined for determining increasing factors of blood viscosity. Dog blood and Urographin (a hypertonic contrast medium) were used; the plasma osmolality was changed by Urografin suspended in blood. The viscosity was measured for normal RBC and glutaraldehyde-treated RBC suspensions with a cell volume concentration. The RBC deformability was evaluated from the difference in viscosity between the two suspensions. It was shown that normal RBC suspension increased the viscosity with increase in osmolality at high shear rate; hardened RBC suspension decreased the viscosity with increase in osmolality. It was concluded that the RBC deformability decreased with increasing osmolality.     

Effects of hemolysis, hematocrit, RBC swelling, and flow rate on light scattering by blood in a 0.26 cm ID transparent tube

The intensity of light scattering by blood in a tube of diameter 0.26 cm was measured with an apparatus devised by us at different angles on an incident cross-sectional plane. Changes in angular distribution of light intensity associated with hemolysis, and changes in hematocrit (Ht), red blood cell (RBC) swelling, and flow rate were plotted on polar coordinates. The dyssymmetry index, defined as the ratio of the intensity of light at 45 degrees to that at 135 degrees, was used to characterize the shape of the diagrams of light scattering. The index decreased with Ht, flow rate, but increased with RBC swelling. It is concluded that light scattering by blood requires intactness of the RBC membrane. Even when the cell membrane is intact, light scattering is subject to changes with the flow rate of blood, presumably due to RBC aggregation.     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase

Various flavonoids were found to be relatively selective inhibitors of arachidonate 5-lipoxygenase which initiates the biosynthesis of leukotrienes with the activity of slow reacting substance of anaphylaxis. Cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone) was most potent, and the enzyme partially purified from rat basophilic leukemia cells was inhibited by 97% at a concentration of 10 microM (IC50, about 0.1 microM). 12-Lipoxygenases from bovine platelets and porcine leukocytes were also inhibited but at higher concentrations (IC50, about 1 microM), and fatty acid cyclooxygenase purified from bovine vesicular gland was scarcely affected. The compound at 10 microM suppressed by 99% the immunological release of slow reacting substance of anaphylaxis from passively sensitized guinea pig lung (IC50, about 0.4 microM).     

New In-vitro Test for IgE-mediated Hypersensitivity in Man

A new simple and sensitive in-vitro method for the diagnosis of type 1 (IgE-mediated) hypersensitivity in man is described. Sliced human skin is passively sensitized by reaginic serum from allergic patients and the presence of antigen-specific IgE on the sensitized slices is detected by assay of antigen-evoked histamine release. Serum from 12 out of 14 patients with clinical respiratory allergy and positive skin tests gave significant antigen-specific histamine release. This method, which is essentially an in-vitro model of the Prausnitz-Küstner reaction, should prove of value in the diagnosis of human reaginic hypersensitivity in man.     

Inactivation of T Antigen-Forming Capacities of Simian Virus 40 and Adenovirus 12 by Ultraviolet Irradiation

Methods to measure T antigen-forming capacities of simian virus 40 (SV40) and adenovirus 12 (Ad12) were investigated, and a method to measure the capacity in terms of T antigen-forming units was employed by the use of cytosine arabinoside. Plaque-forming units and T antigen-forming units of SV40, SV40 deoxyribonucleic acid, or Ad12 were inactivated by ultraviolet (UV) irradiation at the same rate, roughly following a single-hit curve. T-antigen formation by UV-irradiated SV40 and Ad12 was enhanced in cells multiply infected and in cells in a growing state. These observations showed that it was difficult or impossible to estimate the size of the gene for T antigen by UV inactivation.