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Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils.

Changes in ninhydrin positive material, free amino acids and protein content during germination of seeds of Lens culinaris Med. have been studied. Ninhydrin positive material and free amino acids reached their highest concentration at the fifth day of germination. Total protein which represents 21% of the total dry weight of the lentil cotyledons, suffers a degradation of only 24% in seven days of germination; in the same period of time, reserve proteins underwent a degradation of 69%, legumin being the more abundant at the start, and the more rapidly depleted. Five different classes of proteolytic activities have been reported in lentil cotyledons: caseinolytic, active against the reserve proteins of the lentil cotyledons themselves, aminopeptidase, peptidehydrolase, carboxypeptidase and dipeptidase. The removal of the axis did not seem to exert any significant influence on the enzymatic activity.

Amino Acids↗

Separation, isolation and amino acid composition of uremic peptides.

Gel filtration and thin layer chromatography were conducted on sera from uremic patients and normal subjects for the isolation of nitrogenous substances unique to uremia. Many ninhydrin-positive substances were found in greater amounts in uremic patients compared to normal subjects. Some of these ninhydrin-positive substances were also detected by staining with chlorine-tolidine. Amino acid analysis of these substances showed considerable qualitative and quantitative differences, perhaps reflecting interference with enzymatic activity by the uremic environment.

Amino Acids↗

Potential sources of errors in cation-exchange chromatographic measurement of plasma taurine.

We examined the potential sources of error in automated cation-exchange chromatographic quantitation of plasma taurine, both in sample preparation and in the analysis. Principal sources of error include: use of serum instead of plasma, which produces gross overestimates; use of tripotassium ethylenediaminetetraacetate (EDTA) as anticoagulant in systems involving ninhydrin detection (a ninhydrin-positive contaminant of EDTA emerges coincident with taurine); contamination with platelets; and placing volumes exceeding 20 microL on the cartridge used in the Technicon TSM Amino Acid Analyzer. We arrived at a simple technique in which we use EDTA as anticoagulant, micropore filtration to produce platelet-free plasma, and o-phthalaldehyde as the detection reagent for the sensitivity required to measure accurately the low concentration of taurine in plasma.

Autoanalysis↗

Field studies of a reusable polyamide filter for detection of Schistosoma haematobium eggs by urine filtration.

A reusable monofilament polyamide (Nytrel) filter and ninhydrin stained paper filter were compared in syringe filtration of urine to detect Schistosoma haematobium eggs in the urine under field conditions. The sensitivity of the technique using the Nytrel filter was similar to that of ninhydrin stained paper filters. The quantitative geometric mean S. haematobium egg counts obtained with the Nytrel filter were similar to those obtained with the paper filter.

Filtration↗

Identification of a 2-aminotetrose in a lipid-soluble fraction of rat liver.

Chromatography on DEAE-cellulose of chloroform:methanol:water (10:10:3) extracts obtained from the livers of rats pulsed 50 min with [32P]Pi and D-[1-3H]-mannose yielded two radioactive components which eluted at salt concentrations greater than 0.1 M. One of the components yielded, upon mild acid treatment (20 mM HCl, 100 degrees C, 20 min), a low molecular weight water-soluble component (PO), which contained most of the 32P label associated with the parental lipid-oligosaccharide. PO was periodate-negative, ninhydrin-negative, and permanganate-positive. The phosphate group was stable to strong acid, which converted PO to a single, 32P-labeled product, but the phosphate was readily released by incubation under mild alkaline conditions. Strong alkali produced a product reactive with periodate, ninhydrin, and fluorescamine. When dephosphorylated PO was hydrolyzed with acid and the products were converted to the alditol acetate derivatives, gas chromatography revealed a single major component. Gas chromatography/mass spectroscopy of this compound showed it to be the derivative of a 2-acetamido-2-deoxytetrose. That 2-acetamido-2-deoxytetrose is glycosidically bound in PO is indicated by the fact that PO must be hydrolyzed by strong acid before reduction with NaBD4 is able to incorporate deuterium into the tetrose.

Amino Sugars↗

[Excretion of organic compounds bei microorganisms in presence of diphenylamine].

Diphenylamine (DPA) causes an increased leakage of organic compounds with resting cells of Candida guilliermondii, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus already after a treatment of 1 h. The leakage depends both on the concentration of DPA and the duration of the treatment. Moreover, DPA influences the composition of the leaked ninhydrine-positive compounds. After 20 h of treatment an increased release of mannan and ribose is observed in addition to the stronger leakage of ninhydrine positive compounds. With regard to the relation between structure and activity of DPA, the activity of 21 compounds was investigated. The two apolar phenyl groups as well as the secondary amino group being able to form ionic bounds are essential for maximal activity. A disturbance of the function of the cell membrane caused by DPA is discussed.

Acetates↗

[ABO blood grouping of fingerprint by means of immunohistochemical procedure].

For the purpose of ABO-blood typing on fingerprints, the detection of blood group substances in fingerprints attached on nitrocellulose filter or paper was performed immunohistochemically using avidin-biotin-peroxidase complex (ABC) method. At first, it was fundamentally tested whether ABO-blood typing could be specifically performed for the fingerprints of known ABO blood group, being made experimentally on nitrocellulose filter or paper, and the effect of fixation and paper quality for the detection was also examined. And, ABO-typing was carried out using transferred fingerprints from a slide glass to a nitrocellulose filter. Moreover, using a fingerprint of unknown ABO-type, serially repetitive blood typing (three times) was compared to individually performed grouping (three tests) after dividing a single fingerprint into three parts. In addition, the method of serial ABO-blood typing after the morphological detection of fingerprints by ninhydrine was also considered. As results, according to primary antibodies applied, ABH activities were specifically detected in the fingerprints on nitrocellulose filter and paper. For the fixation procedure of very minute blood group substances to paper, heat fixation was the most effective and methanol fixation was also available. The intensity of immunostaining of fingerprints decreased according to the deterioration of paper quality used. And, the transferred fingerprints on nitrocellulose filter were also specifically typed. Serially repetitive blood typing (anti-B-->anti-A-->anti-H) was possible to fingerprints on nitrocellulose filter, but it gave poor results to those on paper. To overcome this difficulty, after being detected morphologically by ninhydrine, iodine or aluminium powder and decolored thereafter, a fingerprint on paper was divided into three parts and specific blood typing was possible. As for the double blind test to fingerprints on paper, 22 out of 35 fingerprints were specifically typed and the rate of correct typing was 62.9%. From these results, it can be considered that this method is available for ABO-typing of fingerprints of secretor type in forensic practice.

ABO Blood-Group System↗

Automated measurement of 13C enrichment in carbon dioxide derived from submicromole quantities of L-(1-13C)-leucine.

The 13C enrichment of the carboxyl carbon of leucine was measured by isotope ratio mass spectrometry after conversion to CO2 by reaction with ninhydrin in a Vacutainer and cryogenic purification using the Finnigan MAT Breath Gas Analysis System designed for processing 13C breath test samples. The sources of error which arise with submicromole samples are examined and corrections provided for suboptimal mass spectrometer signals and contamination of the evolved CO2 with CO2 from the reaction medium. The main limitations to the accuracy and precision of the method are not instrumental but arise from the contamination with residual CO2 in the reaction medium, and this sets a lower limit of around 0.25 mumol leucine on the practical sample size. This is an improvement of about five-fold on the previous manual method of CO2 isolation.

Autoanalysis↗

Fixation of amino acids and oligopeptides by freeze-drying in polyacrylamide gel electrophoresis.

Following polyacrylamide gel electrophoresis in an acetic/formic acid buffer, pH 2.0, in fabric reinforced gels, amino acids and oligopeptides could successfully be fixed in the gel by freeze-drying. Lyophilization of the fabric reinforced polyacrylamide gel after electrophoresis resulted in a dry film which absorbed ninhydrin solution quickly and uniformly, thus improving the detection limit for amino acids and oligopeptides with molecular weights ranging from 189-1045. Most amino acids were detected with a sensitivity of 0.1-0.25 microgram and for oligopeptides the detection limit was found to be 0.5-5 microgram.

Amino Acids↗

Chemical activation of nitrocellulose membranes for peptide antigen-antibody binding studies: direct substitution of the nitrate group with diaminoalkane.

A method to covalently link peptide and proteins, through a diaminoalkane spacer to nitrocellulose membrane was developed for immunochemical applications. Initially the nitrocellulose membrane was modified by covalent incorporation of diaminoalkane spacers without any prior activation. The incorporation was shown primarily to involve alpha-elimination of the nitrate groups, and an imine was formed between the carbonyl group on the membrane and the diaminoalkane. The rate of incorporation increased exponentially with the length of the diaminoalkane as determined by a ninhydrin colorimetric reaction, which was developed for the study. More than 200 nmole diamine per mg nitrocellulose could be incorporated, but less than 11 nmol/mg (63 nmol/cm2) was chosen in order to retain the strength of the membrane. The primary amino groups of the modified membrane was glutaraldehyde activated and the octapeptide, angiotensin II, was covalently bound. A dot immunoassay was performed where specific anti-angiotensin II antibodies reacted with the peptide and was visualized by peroxidase coupled secondary antibodies. The results were quantified by video densitometry above 0.005 microgram AII per cm2. The immunoassay showed improved detection of the peptide on the activated as compared to unactivated membrane as well as increased retention of radiolabeled [125I]angiotensin II.

Angiotensin II↗

Quantitative determination of chitosan and the percentage of free amino groups.

Chitosans were quantified with ninhydrin, a reagent normally used for recognizing and quantifying amino groups. The reaction was time dependent and there was no effect when different acids were added to the mixture. This method was used to determine the percentage of free amino groups in chitosans of different origins.

Amines↗

Biochemical and physiological parameters on the skin surface of healthy test persons: a contribution towards the interpretation of the results obtained by a screening program.

Biochemical and physiological examinations were carried out on the skin surface of a total of 115 healthy persons ranging from the age of 7-79 years. In these investigations the following striking findings were made: 1. There is a significant correlation between the results obtained by the ferricyanide, the phenol sulfuric acid and the ninhydrin methods in the so called water solubles. 2. The relationship between the fraction of wax and cholesterol esters and the squalene fraction in the skin surface lipids influences the release of free fatty acids by bacterial lipases. 3. With regards to most parameters tested there are great differences between the 8-12 year age group and the older age groups. 4. There are significant differences in the 8-12 year age group between the male and female test persons. 5. With regards to the parameters tested there are also significant differences between the various test areas.

Adolescent↗

Acid-labile amino acid precursors in the Murchison meteorite. II: A search for peptides and amino acyl amides.

The basic fraction of the acid-labile amino acid precursors found in hot water extracts of the Murchison meteorite was treated with ninhydrin under conditions that give destruction of free amino acids but allow nearly complete recovery to peptides and amino acylamides. After this treatment and a subsequent acid hydrolysis, the amount of amino acids found corresponded to less than 30 per cent of the precursor content of the basic fraction. Since only 30 percent of the total extract precursors are found in the basic fraction, these results indicate that such compounds, if present, account for no more than 9 mole per cent of the total acid-labile amino acid precursors of the meteorite hot water extract.

Amides↗

Diversity of the polar lipids of the food-borne pathogen Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive bacterium that can adapt to high salinity and cold. Because the membrane lipids may play a role in its survival and adaptation, we have examined the polar lipids of L. monocytogenes. Extraction of total lipids from L. monocytogenes yielded 7 +/- 1 mg/mL wet cells. Polar lipids represented 64% of total lipids and contained 9% lipid-phosphorus. Polar lipids were separated into 14 components by two-dimensional thin-layer chromatography. Eight components (88% of polar lipids) contained lipid-phosphorus; among these was one major component (34% of polar lipids). Two other phospholipids were ninhydrin-positive components and accounted for 15% of the polar lipids. Orcinol staining revealed two glyco- or sulfo-lipids accounting for 9% of polar lipids. Five components (4% of polar lipids) were amino components free of phosphorus. The major component contained 46% of its fatty acids as 15:0 anteiso, 24% as 17: 0 anteiso, and 11% as 15:0 iso. The fatty acid profile of the remaining polar lipids was variable, consisting primarily of 16:0, 18:0, 15:0 anteiso, and 17:0 anteiso. Their unsaturation level was < or = 20%; however, the major phosphoaminolipid component was 46% unsaturated. The ratios of 15:0 anteiso/17:0 anteiso and 15:0 anteiso/15:0 iso were similar in all classes, averaging 1.5 and 4.5, respectively. Since the adaptation process to stressful environments involves activation of a membrane transport system for the protectant glycine betaine, the membrane lipids may play a role in enabling transport.

Chromatography, High Pressure Liquid↗

The effect of heat shock on amino acid transport and cell volume in 3T3 cells.

In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44 degrees C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.

3T3 Cells↗

A spectrophotometric rate assay of aminoacylase.

Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.

Alanine Dehydrogenase↗

Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization.

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.

Animals↗

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site.

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.

Amino Acids↗