An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides.

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.     

A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays

A simplified method for inorganic phosphate determination has been developed. The method is sensitive, easy, economic, and applicable for estimation of phosphate released in both enzymatic and nonenzymatic reactions. A mixture of hydrazine sulfate and ascorbic acid was used as the reducing agent and the conditions for the development of the molybdenum blue color were optimized. Thus in the 4.0 ml assay system, 0.4 ml of the reducing agent solution containing 20 mg each of hydrazine sulfate and ascorbic acid per milliliter of 1.0 N H2SO4 gave a rapid optimum color development with absorption maximum at 820 nm. Color development showed a linear relationship up to 10 microg Pi concentration. Thus the method has a 2.5x higher range of Pi estimation than that of the Bartlett method. The molar extinction coefficient at 820 nm was higher than that obtained in the Bartlett procedure. Also the molybdenum blue color formed was stable up to 24 h. Under the standard assay conditions, interference from acid-labile phosphate as in the case of Na+,K+ ATPase was at the minimum. The applicability of the method for assay of microsomal Na+,K+ ATPase and glucose-6-phosphatase was checked in microassays (final volume 0.1 ml) in comparison to the conventional procedures which use 3-4 times higher volumes. Likewise the applicability of the method for phospholipid analysis was compared with that of the conventional Bartlett method. Under both test systems the results obtained by the micromethod were identical to those obtained by the conventional methods. In general the method, which rapidly produces quantitatively molybdenum blue color, not only is rapid economical, and convenient but also has wide applicability.     

A simple activity staining protocol for lipases and esterases

A simple activity staining protocol for rapid detection and differentiation of lipases and esterases was developed based on pH drop due to fatty acids released following lipolysis. Though the detection of lipolysis as a function of drop in pH is not new, the present method has been made more sensitive by the judicious selection of the initial pH of the chromogenic substrate, which has been set near the end point of the dye so that even a slight drop in pH results in immediate color change. In the present case, the dye phenol red was taken, which has the end point at pH 7.3–7.4 where the color is pink. A slight drop due to fatty acid release results in yellow coloration. The assay has high reproducibility and can detect as low as 0.5 p-NPP enzyme units within 15 min. In addition, this method can be used for various lipidic substrates such as oils and tributyrin, making it suitable for both lipases and esterases.     

Bombesin microinjected into the dorsal vagal complex inhibits vagally stimulated gastric acid secretion in the rat

Medullary sites of action for bombesin-induced inhibition of gastric acid secretion were investigated in urethane-anesthetized rats with gastric fistula. Unilateral microinjection of bombesin or vehicle into the dorsal vagal complex was performed using a glass micropipet and pressure ejection of 100 nl volume; gastric acid output was measured every 10 min by flushing the stomach. Microinjection of vehicle into the dorsal vagal complex did not alter gastric acid secretion (1.9 +/- mumol/10) from preinjection levels (2.9 +/- 0.8 mumol/10 min). Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, at a 77 pmol dose into the dorsal vagal complex stimulated gastric acid secretion for 100 min with a peak response at 40 min (24.1 +/- 3.2 mumol/10 min). Concomitant microinjection of RX 77368 (77 pmol) with bombesin (0.6-6.2 pmol) into the dorsal vagal complex dose dependently inhibited by 35-86% the gastric acid response to the TRH analog. Bombesin (6.2 pmol) microinjected into the dorsal vagal complex inhibited by 17% pentagastrin infusion-induced stimulation of gastric acid secretion (13.2 +/- 0.8 mumol/10 min) whereas intracisternal injection induced a 69% inhibition of the pentagastrin response. These results demonstrate that the dorsal motor complex is a sensitive site of action for bombesin-induced inhibition of vagally stimulated gastric secretion. However, other medullary sites must be involved in mediating the inhibitory effect of intracisternal bombesin on pentagastrin-stimulated gastric acid secretion.     

Non-hydroperoxy-type Peroxides as Autocatalysts of Lipid Autoxidation

In this paper, we present a simple and sensitive method for the assay of intracellular neutral lipids of the yeast, Lipomyces starkeyi. In this method, we used a tower-shaped mixer for cell disruption and applied a diagnostic triglycerides assay kit as a sensitive color developer. This mixer could treat 14 samples at one time. No effect of pH on cell disruption was detected within the pH range of 1.6 to 6.8. Twenty min was chosen for cell disruption and for homogeneous dispersion of intracellular neutral lipids. The analytical results agreed well with those obtained by three conventional methods. The new method is simpler and of good reliability.     

Plasma carnitine and acetyl-carnitine levels at different times of the day

An interest in both biochemical and clinical carnitine investigation has recently developed. A more complete and extensive study is obtained if acetyl-carnitine as well as carnitine are investigated. This research, using an improved and simplified method for carnitine and acetyl-carnitine determination in the same sample (1 ml) without radioisotopic tracer use, investigates if there are the same differences in their plasma levels at different times of the day. The sample was eluted in a chromatographic column (55 X 15 mm) containing Sephadex G-25M with phosphate buffer (25 mmol/l, pH 7.4). The fraction containing acetyl and free carnitine was divided and employed separately for two assays. The carnitine assay uses an enzymatic reaction catalyzed by carnitine acetyl-transferase (CAT) and measurements are carried out spectrophotometrically. The calibration curve shows r = 0.987 and sensitivity at 5 mumol/l (reference plasma values: 38 +/- 3 mumol/l in 9 subjects). The acetyl-carnitine assay is carried out concentrating the sample by lyophilization and then measuring the enzymatic coupled reactions catalyzed by CAT, malate dehydrogenase and citrate synthase fluorimetrically. The calibration curve gives r = 0.991 and sensitivity at 1.4 mumol/l (reference plasma values: 2.8 +/- 0.3 mumol/l in 9 subjects). Both assay methods are measured at the end point. The carnitine and acetyl-carnitine measured in the plasma of 6 normal subjects at different times of the day vary respectively from 28 to 37 mumol/l and from 1.1 to 5.2 mumol/l in agreement with plasma free fatty acid (FFA) variation from 230 to 779 microEq/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A.

A novel radiochemical assay for long-chain fatty acid:CoASH ligase activity (AMP) (EC 6.2.1.3) has been developed based on the conversion of [3H]CoASH to long-chain fatty acyl CoA. Fatty acyl [3H]CoA was quantitatively retained on Millipore filters upon filtration of the acidified reaction mixture under conditions where the [3H]CoASH was not retained. The assay was developed using microsomes derived from isolated fat cells as the source of fatty acid:CoASH ligase activity. The assay performed at 25 degrees C for 10 min was linear with added microsomal protein up to 7 mug. The assay was linear with time up to 24 min when 1 mug of protein was employed. Fatty acid:CoASH ligase activity was strongly dependent on ATP and magnesium, was stimulated by Triton WR-1339, and was two- to fivefold dependent on added fatty acid. The filter assay is easier than existing assays based on incorporation of labeled fatty acid and is equally sensitive.     

Purification and properties of guinea-pig submandibular-gland kallikrein.

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.     

Specific enzymatic assay for D-glucarate in human serum

A sensitive and specific spectrophotometric assay was developed to determine levels of D-glucarate in human serum. This assay makes use of the Escherichia coli glucarate catabolic enzymes D-glucarate dehydrase, alpha-keto-beta-deoxy-D-glucarate aldolase, and tartronate semialdehyde (TSA) reductase, to convert D-glucarate to equimolar quantities of pyruvate and TSA. In a one-tube reaction that included NADH, lactate dehydrogenase, and the three E. coli enzymes, 1 mumol of D-glucarate was quantitatively converted to 1 mumol each of D-glycerate and L-lactate with concomitant utilization of 2 mumol of NADH. Using this method, D-glucarate in serum was measured, along with quantitative recovery of authentic D-glucarate from duplicate serum samples to which it had been added. Glucarate is a major serum organic acid, approximating blood pyruvate levels previously determined by others.     

Proton transport catalyzed by the sodium pump. Ouabain-sensitive ATPase activity and the phosphorylation of Na,K-ATPase in the absence of sodium ions

The possibility that H+ might substitute for Na+ at Na+ sites of Na+,K+-ATPase was studied. Na+,K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+,K+-ATPase). The specific activity was 1.1 mumol Pi/mg/min at pH 5.7, whereas the specific activity of Na+,K+-ATPase was 14 mumol Pi/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+,K+-ATPase was inhibited as effectively as Na+,K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+,K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.     

Spectrophotometric assay of hydroxylated by-products in penicillin V fermentations and its application in screening of mutant penicillin producer strains on agar media

A fast and sensitive spectrophotometric method has been developed to measure the level of hydroxylated by-products (p-hydroxyphenoxyacetic acid and p-hydroxypenicillin V) in penicillin V fermentations. The method is based on a color reaction of the above-mentioned phenolic by-products with nitrous acid, yielding yellow nitroso derivatives. Both the nitroso derivative of p-hydroxyphenoxyacetic acid and that of p-hydroxypenicillin V have an absorption peak at 450 nm in alkaline solution with a molar absorption of 4.00 x 10(3) M-1cm-1 for both compounds. No fermentation medium components were found to interfere considerably with the assay. On the basis of the color reaction, an agar prescreen method has been developed for isolation of nonhydroxylating Penicillium chrysogenum strains in strain-improvement programs.     

High-performance liquid chromatography-based assay for the quantitation of protein maleylation at the picomole level

A new HPLC-based assay for the direct determination of the extent of maleylation of proteins is described. The rationale is founded on the HPLC detection of maleic acid that results from cleavage of maleyl lysyls by low pH treatment. The method, employing a C-18 reverse-phase column eluted isocratically with 0.01 M acetic acid, pH 5.4, and absorbance monitored at 210 nm, is sensitive (lower limit 1 pmol) and linear in the range 1 pmol-0.5 mumol and allows recovery of the native protein.     

Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips

Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G–horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.     

The effect of neurotensin and secretin on gastric acid secretion and mucosal blood flow in man

Neurotensin stimulates pancreatic secretion directly and by potentiating the effect of secretin. Neurotensin also inhibits gastric secretion. Secretin inhibits gastric secretion as well, but whether it also interacts with neurotensin is not known. Secretin is known to inhibit gastric mucosal blood flow (GMBF). The effect of neurotensin on GMBF is not known. Acid secretion (triple lumen perfused orogastric tube) and GMBF ([14C]aminopyrine clearance) were therefore measured in 6 subjects during neurotensin, secretin and neurotensin plus secretin infusions. Neurotensin plus secretin reduced acid secretion by a median 130 (range 34-394) mumol/min which was significantly greater than either neurotensin at 36 (7-67) mumol/min or secretin 54 (20-347) mumol/min alone (P less than 0.05). This effect appeared independent of GMBF. Neurotensin plus secretin reduced GMBF by 14 (12-27) ml/min but not significantly more than neurotensin at 11 (3-20) ml/min or secretin 18 (2-27) ml/min alone. Further, there was no correlation between changes in acid output and GMBF during infusion of the peptides. We conclude that the inhibitory effects of neurotensin and secretin on gastric secretion are at least additive and together they may function as an 'enterogastrone'.     

Interference of biogenic amines with the measurement of proteins using bicinchoninic acid

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.     

L-ascorbic acid 2-sulphate. A substrate for mammalian arylsulphatases.

Ascorbic acid 2-sulphate has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates. At its optimum pH of 4.8 sulphatase A(aryl-sulphate sulphohydrolase EC 3.1.6.1.) hydrolyses ascorbic acid sulphate with a specific activity of 90 mumol/mg per min (150 mumol/mg per min with nitrocatechol sulphate at pH 5.6). At pH 4.8 the kinetics are non-Michaelis. At pH 5.6 Michaelis kinetics are obeyed and Km 12 21 mM ascorbic acid 2-sulphate. K2SO4 is a competitive inhibitor with a Ki of 0.2 and 0.6 mM at pH 4.8 and 5.6, respectively. Sulphatase A is converted into a substrate-modified form during its hydrolysis of ascorbic acid sulphate. Sulphatase B also hydrolyses ascorbic acid 2-sulphate. At pH 4.8 and in the presence of 0.15 M NaCl the specific activity is 0.92 mumol/mg per min (90 mumol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. Km is 8 mM. K2SO4 is a competitive inhibitor with a Ki of 0.1 mM. Ascorbic acid is not hydrolysed at a detectable rate by the arylsulphatases of the mollusc Dicathais orbita or of Aerobacter aerogenes.?     

Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

A Colorimetric Assay of Lipoyl-N-?-Lysine Hydrolysis Activity Using 2,6-Dibromoquinone-4-Chlorimide

Lipoic acid is a coenzyme for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched chain-ketoacid dehydrogenase, and the glycine cleavage system. Lipoic acid is covalently attached through an amide to the ?-amino group of specific lysine residues of these enzymes. Lipoamidase hydrolyzes the amide bond of lipoyl-N-?-lysine. Because of the difficulty in quantitating lipoic acid or lysine released by hydrolysis of lipoyl-N-?-lysine, a sensitive assay of lipoamidase activity was developed based on quantitation of lipoic acid liberated from lipoyl-?-lysine using 2,6-dibromoquinone-4-chlorimide (DBQC). This method involves acidification of the assay mixture with HCl and separation of lipoic acid from lipoyl-N-?-lysine by extraction into ethyl acetate where it can react with DBQC. This method is as sensitive as methods based on the reaction of lipoic acid with dinitrothiobenzoate and requires only a single extraction, but does not require reduction of the disulfide and the color reagent does not need to be prepared daily. Results obtained using this assay to quantitate lipoic acid released from lipoyl-N-p-aminobenzoate correlated excellently with results obtained using the Marshall–Bratton reaction to quantitatep-aminobenzoate. We have detected lipoyl-N-?-lysine hydrolysis activity that is distinct from that of biotinidase and bile salt-stimulated lipase in lymphoblasts from a patient with biotinidase deficiency. This assay can be used to measure lipoyl-N-?-lysine hydrolysis activity in tissues, especially those with little or no biotinidase activity.     

An improved micromethod for tyrosine estimation

A modified and improved micromethod for tyrosine determination has been developed. The method is sensitive, economic and applicable for estimation of tyrosine released in enzymatic reactions and in tissue. A range of Folin-Ciocalteu (FC) reagent was used to optimize the conditions for the development of blue color. Thus in 1.5 ml of the assay system, the suitably diluted FC reagent at the final concentration of 0.2 N gave a rapid optimum color development with an absorption maximum at 750 nm. Color development showed a linear relationship in the range of 2 to 16 microg tyrosine for a described assay system under optimized conditions. Thus, the method is 3-fold more sensitive in terms of its estimation range than a conventional method. The blue color formed was stable up to 24 h. The applicability of the method for tyrosine determination in the assay of lysosomal cathepsin D and in tissue was checked by comparison to the conventional procedure. Under both systems the results obtained by the micromethod were identical to those obtained by the conventional method. In general the method that produces quantitatively a blue color, not only is rapid and economical in terms of chemical usage but also has application for routine biochemical analysis.     

Colorimetric micromethods for glutaraldehyde determination by means of phenol and sulfuric acid or phenol and perchloric acid

Aqueous micromolar solutions of glutaraldehyde produce a yellow color when treated with 20% phenol in ethanol followed by concentrated sulfuric acid or with phenol in 70% perchloric acid. The absorbance at 482 or 479 nm, respectively, is linearly related to the glutaraldehyde concentration. Of the two methods developed, the sulfuric acid-phenol assay gives a higher sensitivity and the perchloric acid-phenol assay allows the determination of glutaraldehyde in the presence of sugars and proteins.