Reaction Rate Assay Research Articles

Large Domain Formation in POPC/POPE Lipid Bilayers at the Maximum Solubility Limit of Cholesterol

Lipid bilayers composed of POPC and POPE usually stay as 1-phase mixtures at room temperature, with or without incorporation of modest amount of cholesterol. However, at the maximum solubility limit of cholesterol, possibility of domain formation has been suggested based on the detail molecular packing patterns of phospholipids with cholesterol. The maximum solubility of cholesterol in a lipid bilayer is the highest mole fraction of cholesterol that can be incorporated into a lipid bilayer before cholesterol crystals precipitate. Previously, the maximum solubility of cholesterol in mixtures of POPC/POPE lipid bilayers has been measured using a cholesterol oxidase (COD) reaction rate assay. The maximum solubility of cholesterol was determined to be 67 mol % in POPC bilayers and 50 mol % in POPE bilayers. In mixtures of POPE/POPC, the maximum solubility of cholesterol increases linearly as a function of the ratio POPC/(POPE+POPC). In this study, planar lipid bilayers of POPC and POPE at their cholesterol solubility limits were incubated on mica surfaces inside hydrated chambers. Although the bilayers appeared having uniform fluorescence intensity initially, we found that large lipid domains became visible by fluorescence microscopy after several days of incubation. Previously, it has been suggested that cholesterol may form a “hexagonal” regular distribution pattern at the maximum solubility limit in POPE bilayers and a “maze” pattern at the maximum solubility in POPC bilayers. Thus, it is possible that the lipid mixtures separated into POPC-rich and POPE-rich lipid domains, each having their signature lipid packing pattern and mole fraction of cholesterol.

The Maximum Solubility of Cholesterol in POPC/POPE Lipid Mixtures

Cholesterol is a major constituent of cell membranes and has many important cell functions. The maximum solubility of cholesterol in a lipid bilayer is the highest mole fraction of cholesterol that can be incorporated into a lipid bilayer before cholesterol crystals precipitate. The maximum solubility can provide valuable information about cholesterol-phospholipid interaction. In this study, the maximum solubility of cholesterol in mixtures of POPE/POPC lipid bilayer has been investigated systematically using a cholesterol oxidase (COD) reaction rate assay.

Resveratrol ameliorates hepatic injury via the mitochondrial pathway in rats with severe acute pancreatitis

To gain insight into the processes by which severe acute pancreatitis induced apoptosis takes place in the liver, and to observe the protective effect of resveratrol on hepatic injury, a rat model of severe acute pancreatitis was induced by administering 4% sodium taurocholate through the common biliopancreatic duct. Pancreatic and hepatic injury was assessed by histology. Serum ALT (alanine aminotransferase), AST (aspartate aminotransferase) and total bilirubin were determined by reaction rate assay, and the serum levels of TNF-α (tumor necrosis factor-alpha) and IL-6 (interleukin-6) were detected by ELISA (enzyme linked immunosorbent assay). We investigated cytochrome c released from mitochondria and used the RT-PCR (reverse transcription PCR), Western blot technique to evaluate Bax, Bcl-2, and caspase-3 expression levels in hepatic tissue over the time course of apoptosis. Changes in hepatic cell mitochondrial membrane potential were observed by confocal laser scanning microscopy. The majority of cytochrome c release occurred early in apoptosis from mitochondria, which undergo gradual hepatic impairment. The released cytochrome c can be reduced by resveratrol through both up-regulation of Bcl-2 and down-regulation of Bax and caspase-3. These data provide substantial evidence that apoptosis is involved in hepatic injury during the severe acute pancreatitis process and that resveratrol can ameliorate the situation, thus protecting liver function in rats with severe acute pancreatitis.

Ceramide Drives Cholesterol Out of the Ordered Lipid Bilayer Phase into the Crystal Phase in 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Cholesterol/Ceramide Ternary Mixtures

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.

Purification and properties of urease from Brevibacterium ammoniagenes.

Urease was purified to a homogeneous state from cell-free extracts of a strain of Brevibacterium ammoniagenes. The enzyme was found to have a molecular weight of 200,000, to consist of three identical subunits with a molecular weight of 67,000, and to have an isoelectric point at pH 4.1. The amino acid profile was similar to that of jack bean urease. Methionine and leucine were shown to be the N- and C-terminal residues, respectively. The enzyme contained 1 atom of nickel per subunit. The enzyme was most active at about pH 7 and at 65°C, and was stable betweeen pH 7 and 10 (at 30°C for 5 hr) and below 50°C (at pH 7.5 for l0min). Among the compounds examined, Hg2+, Cu2+, PCMB, and hydroxyurea inhibited the enzyme strongly. The Km value of enzyme for urea was found to vary from 18 to 72 mm depending on pH and the type of buffer used. It was proved that the enzyme can be used for the determination of urea by the reaction rate assay method.

Enzymic reaction-rate assay for urinary creatinine.

Enzymic reaction-rate assay for urinary creatinine.

The assay of arylesterase in serum using two new colorimetric substrates, ω-nitrostyrylacetate and propionate

1. (1) The synthesis of 4-acetoxy-3-methoxy-ω-nitrostyrene and 4-propionyloxy-3-methoxy-ω-nitrostyrene as substrates for the assay of arylesterase activity is described. These substrates are stable when stored in the dark for at least one year. 2. (2) Serum isolated from normal males and females aged between 25 and 35 years was used as a source of esterase activity. 3. (3) The chromophore (4-hydroxy-3-methoxy-ω-nitrostyrene) absorbs at 505 nm and has a molar extinction coefficient of 26800 in sodium carbonate/bicarbonate buffer, pH 9.5. 4. (4) Both acetate and propionate substrates are more soluble in aqueous alcohol than in water or buffer alone and the substrates were therefore prepared in 10% methanol. Since some non-enzymic hydrolysis occurs at 37°C, assays were carried out at 30° C. Details of both single point and reaction rate assay procedures are described. 5. (5) The properties of the arylesterases were determined in human serum using the single point and reaction rate assays. The data are similar to those previously described for these esterases using well-established procedures. 6. (6) The assays described using the ω-nitrostyryl acetate and propionate substrates are simple to perform and can be readily adapted for use in the clinical chemistry laboratory. Large numbers of samples can be rapidly assayed with the minimum number of manipulative steps.

生化学自動分析機 Clinalyzer JCA-5KR による CPK 活性の反応速度測定法に関する研究

生化学自動分析機 Clinalyzer JCA-5KR による CPK 活性の反応速度測定法に関する研究

The determination of γ-glutamyl transpeptidase by reaction rate assay at 37° C

The determination of gamma-glutamyl transpeptidase by a reaction rate assay using optimal reaction conditions at 37 degrees is described. Specific conditions and instrument settings are described for the LKB Reaction Rate Analyser but the actual assay conditions are applicable to any similar reaction rate system. The precision of the method has been evaluated and a reference range for normal male (less than 45 U/l) and female (less than 30 U/l) subjects determined.

An Initial Reaction Rate Assay for "Glycerate Dehydrogenase"

Abstract Concentrations of substrate, coenzyme, and hydrogen ion, optimal for the reduction of hydroxypyruvate at 37°C by normal and pathological human sera are described. We confirmed, by using the supernatant fluid from homogenized human heart and liver tissue, that the faster-moving isoenzymes have a greater affinity for substrate and coenzyme than the slowermoving isoenzymes, which require higher concentrations for saturation. LDHx, present in human seminal plasma, appears to have the lowest Michaelis constant for hydroxypyruvate. Glycerate dehydrogenase was found to be stable if the standard assay temperature was increased from 25° to 37°C.