Enhanced enzymatic activity and stability of trypsin by reductive alkylation in solid phase

Amino groups of trypsin (EC 3.4.21.4) were reductively alkylated in solid phase to obtain a surface-active and biologically active enzyme in an o/w emulsion system. Trypsin adsorbed on a benzamidine-sepharose column was reductively alkylated with n-octanal in the presence of sodium borohydride, i.e., trypsin-C8. Activity of trypsin-C8 against Nalpha-benzoyl-L-arginine-p-nitroanilide was three times higher than that of native trypsin. Activities of trypsin and trypsin-C8 against casein were almost the same. After incubating the trypsin solution at 40 degrees C for 1 h, residual activities in the emulsion and solution systems were 64.2 and 57.4%, respectively. On the other hand, residual activities of native trypsin following incubation were 21.8% in the emulsion system and 33.2% in the solution system. Enhancement of trypsin-C8 stability in the emulsion system may derive from interaction between the hydrophobic areas of trypsin-C8 molecules and the hydrophobic phase of the emulsion.     

Changes in Chlorophyll a and b Content in Dark-Incubated Cotyledons Excised from Illuminated Seedlings: THE EFFECT OF CALCIUM

Cucumber seedlings were illuminated for various time periods, cotyledons excised, placed in the dark, and changes in chlorophyll a and b content monitored. During the dark periods, chlorophyll b content decreased while chlorophyll a did not. When the illumination time was lengthened, the percentage of chlorophyll b decomposition from initial levels decreased. Ca²⁺ at 50 millimolar prevented the decrease in chlorophyll b and caused a decrease in chlorophyll a. The effect of Ca²⁺ decreased with increased illumination time. Cycloheximide and chloramphenicol inhibited chlorophyll b decrease, but did not induce chlorophyll a decrease.     

Comparison of renal ornithine transcarbamylase activities within different chicken breeds

Comparisons were made of the renal ornithine transcarbamylase (OTC) activities within different groups of chickens including Japanese native breeds. OTC activities varied markedly within these groups. The Cochin Bantam breed and White Leghorn B line had an especially high activity, about 400 units/g of kidney, in contrast to two Japanese native breeds, Japanese Game (white variety) and Banshuu Gashiwa, and the California Gray breed, which showed a very low activity, the values being almost undetectable. In crossing experiments using the California Gray breed as a tester strain, Cochin Bantam OTC represents a simple autosomal incompletely dominant trait similar to the White Leghorn B line OTC. Kinetic studies using partially purified OTC preparations from the White Leghorn B line and Cochin Bantam breed revealed that both enzymes were identical for a variety of enzymic characteristics. In light of these results, the physiological significance of chick kidney OTC is discussed.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Immunological cross-reactivity of gamma-glutamyltranspeptidases from human and rat kidney, liver, and bile

Antisera to purified gamma-glutamyltranspeptidase (gamma GTP) from human and rat kidney were prepared, and their reactivities toward purified gamma GTP from kidney, liver, and bile were tested. The following results were obtained: 1. On double immunodiffusion, Triton-solubilized gamma GTP, and papain-solubilized gamma GTP from rat kidney gave single precipitin lines which fused completely against antiserum to the purified enzyme from rat kidney. 2. An antigen-antibody complex of human kidney gamma GTP retained about 50% of the catalytic activity of the antigen. 3. Double immunodiffusion showed that the enzymes from human liver, kidney, and bile were immunologically identical. 4. Antiserum to rat kidney gamma GTP partially cross reacted with human gamma GTP, but antiserum to human gamma GTP reacted only very weakly with rat gamma GTP. It is concluded that gamma GTP of human liver, kidney, and bile are immunologically identical and that rat gamma GTP and human gamma GTP have certain antigenic determinants in common.     

Limited autolysis of Ca2+-activated neutral protease (CANP) changes its sensitivity to Ca2+ ions

Ca2+-activated neutral protease (CANP) usually requires mM Ca2+ for activation. The sensitivity of CANP to Ca2+ is greatly enhanced by passing it through a casein-Sepharose column in the presence of Ca2+ ions. This conversion is ascribed to autolysis of CANP. The converted enzyme required 40 microM Ca2+ for 50% activation. Various properties of the converted enzyme were very similar to those of CANP-I, recently found in canine heart muscle. Names of "m-CANP" and "mu-CANP" are proposed for CANPs which require mM and microM order Ca2+ for inactivation, respectively.     

On the electromotor neurons of both electric organs of Pollimyrus isidori (Mormyridae, Teleostei)

The electromotor neurons (EMNs) of the adult electric organ of Pollimyrus isidori and the "giant neurons" (GNs) found in the more rostral part of the spinal cord were investigated both with light and electron microscopical methods. The two kinds of neuron are more or less similar in size, about 30 microns, and are found in the more dorsal part of the spinal cord. The EMNs show somatosomatic gap junctions, as do the GNs. In addition, chemical synapses could be found between nerve fibres and both types of neurons. Histochemical investigations show that the EMNs and the GNs are both AChE positive. On the basis of the arguments presented in the discussion, we believe that the "giant neurons" are the electromotor neurons of the larval electric organ of Pollimyrus isidori.