Role of water receptor in the frog

To elucidate the role of the water receptor in the frog (Rana catesbeiana), reflex activities elicited by its excitation were studied. Application of tap water to the oral mucosa depressed the rhythmical movement of gorge (buccal) respiration, accompanied by an elevation of the inner pressure of the oral cavity (buccal pressure). Tonic reflex discharges were elicited in the nerves innervating the submental and submaxillary muscles, which close the nostrils, the pterygoid and the profound portion of the major masseter muscles, which produce a strong bite, and the geniohyoid and hyoglossus muscles, which elevate buccal pressure. These muscles, except for the pterygoid, also participate in the rhythmical movement of gorge respiration as expiratory muscles. Rhythmical movements in the minor masseter and sternohyoid muscles, which act as inspiratory muscles in gorge respiration, were depressed by the water stimulation of the oral mucosa. These findings indicate that the water receptor plays a role in the interruption of gorge respiratory movements, accompanied by an elevation of buccal pressure.     

Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me₂Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me₂Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me₂Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Cyanopeptolin S, a sulfate-containing depsipeptide from a water bloom of Microcystis sp.

Abstract A new sulfated, cyclic depsipeptide, called cyanopeptolin S, from Microcystis sp. was isolated from a water bloom in the Auensee/Leipzig (Germany). The depsipeptide had a relative molecular mass of 925 and contained l-arginine, l-threonine, l-isoleucine, N-methyl-l-phenylalanine, a l-glutamic acid-δ-aldehyde ring system and a sulfated d-configurated glyceric acid as a side chain. The structure was elucidated by means of two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectroscopy, Fourier transformed infrared spectroscopy and combined gas-liquid chromatography/mass spectrometry. Cyanopeptolin S inhibited trypsin with an IC₅₀≤ 0.2 μg ml⁻¹.     

Structural and systematic study of an unusual tracheid type in cacti

Wide-band tracheids are a specialized tracheid type in which an annular or helical secondary wall projects deeply into the cell lumen. They are short, wide and spindle-shaped, and their bandlike secondary walls cover little of the primary wall, leaving most of it available for water diffusion. Wide-band tracheids appear to store and conduct water while preventing the spread of embolisms. They may be the most abundant tracheary element in the xylem, but they are always accompanied by at least a few vessels. Typically, fibers are absent wherever wide-band tracheids are present. Wide-band tracheids occur in the primary and secondary xylem of succulent stems, leaves and roots in genera of all three subfamilies of Cactaceae but were not found in the relictual genusPereskia, which lacks succulent tissues. In the large subfamily Cactoideae, wide-band tracheids occur only in derived members, and wide-band tracheids of North American Cactoideae are narrower and are aligned in a more orderly radial pattern than those of South American Cactoideae. Wide-band tracheids probably arose at least three times in Cactaceae.     

Short communication: Purification and properties of a glucose-forming amylase of Lactobacillus brevis

An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K _m value for starch was 0.27 mg ml^-1 and its M _r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca²⁺, Mg²⁺, Na⁺ or K⁺ and inhibited by EDTA, KCN, citric acid and l-cysteine.     

人脑神经元特异性烯醇化酶的纯化方法

采用改良的Grace层析方法,经一次DEAE-Sephadex A50柱层析即从人脑中纯化了神经元特异性烯醇化酶,比活力为92.1U/mg,纯化倍数为59.4.该酶纯化后,经SDS-聚丙烯酰胺凝胶电泳鉴定为单一蛋白质谱带.此外,还测定了其部分理化性质,其亚单位分子量为45000,等电点pI为4.7,氨基酸组成分析表明其为一种酸性蛋白质;对2-磷酸甘油酸的K_m值为5.6×10^-4mol/L.     

Firefly luciferase: Purification and immobilization

The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined.     

Interrelationships between water and cellular metabolism inArtemia cysts

Cysts of the crustaceanArtemia are a useful model for studies on intracellular water because they are capable of essentially complete and reversible desiccation. We have used a variety of techniques on this system, the present work being an attempt to estimate the density of intracellular water (ρ_w). The density of individual cysts was evaluated from sedimentation velocity. Heptane displacement methods were used to determine the volume of a known mass of cysts, from which the density was calculated. The two methods produce comparable results. It was shown that the densities and water contents of large masses of cysts accurately reflect those of individual cysts. Cyst densities (ρ_c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (W _f), and temperature dependence was measured for 0.61W _f over 2–41°C. The following refer to 25°C. No marked change was detected in ρ_c until the water content exceeded 0.15W _f, at which ρ_c decreased as a linear function of Wf to maximum water content. However, the cyst does not behave ideally in the sense that the densities of the nonaqueous components and added water are not additive as a function ofW _f. The partial specific volume of water in cysts at maximum hydration was estimated to be 3% larger than that of pure water. These observations are compared with density measurements on other systems, and with previous findings on the physical properties of water in this system.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Precipitation of collagens by polyethylene glycols

Types I, II, and III collagens are readily precipitated at neutral pH by polyethylene glycols (PEG). As the molecular weight fraction of the polyethylene glycols increases, they become more effective as precipitants on a weight basis. The amount of PEG required for precipitation depends on the pH, the ionic strength, and the nature of the buffer or salts present. In tissue culture media, low concentrations of collagens and procollagens may be quantitatively precipitated and readily collected by low-speed centrifugation. Polyethylene glycol precipitation can be used to obtain collagens and procollagens from tissue culture media at either analytical or preparative scale, and since the polyethylene glycols do not bind to collagens, the precipitates may be further analyzed directly by chromatographic or electrophoretic methods.