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PubMed1974

The purification and specificity of a neutral endopeptidase from rabbit kidney brush border.

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.

Albumins↗
PubMed1974

A fluorimetric method for the determination of tryptophan in animal tissues.

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390-400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05-34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56+/-0.76 (s.e.m.) nmol/g wet wt.

Acetates↗
PubMed1975

Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans.

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide "map". 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.

Amino Acids↗
PubMed1977

A second activation peptide from bovine cationic trypsinogen.

1. Although only one activation peptide of bovine cationic trypsinogen has been reported previously, the peptide fraction obtained from activation mixtures shows several bands on paper electrophoresis at pH 6.5. 2. The major band was the peptide previously described. The band second in intensity of staining with ninhydrin (10-20% of that of the main band, as judged by eye) had an electrophoretic mobility consistent with its being related to the main peptide. It appeared on activation both of bulk commercial samples of trypsinogen and, as the Appendix shows, of samples prepared from pancreases obtained at the local abattoir. 3. The second peptide proved to be Phe-Pro-Val-Asp-Asp-Asp-Asp-Lys, and we conclude that it is another activation peptide. We discuss briefly the genetic and phylogenetic implications of our findings.

Amino Acid Sequence↗
PubMed1980

Isolation, characterization and seasonal variations in the concentration of N-epsilon-(gamma-glutamyl)-lysine isodipeptide in the blood plasma of the winter flounder (Pseudopleuronectes americanus).

A ninhydrin-positive compound was isolated from the plasma of the winter flounder (Pseudopleuronectes americanus) and identified as the isodipeptide N-epsilon-(gamma-glutamyl)-lysine. Wide seasonal variations in plasma concentration of this compound are found in the male flounder, whereas it is present only in trace amounts in the plasma of the female flounder throughout the year. In the male flounder the plasma concentration of N-epsilon-(gamma-glutamyl)-lysine rises from January to a peak in May and June. It is during these latter months that the flounder spawn. After spawning, the plasma concentration of the isodipeptide decreases to low basal values in October. These changes in plasma concentration of N-epsilon-(gamma-glutamyl)-lysine may be related to changes in testes weight, spermatogenesis and spawning activity of the winter flounder.

Amino Acids↗
PubMed1980

N6-Trimethyl-lysine metabolism. Structural identification of the metabolite 3-hydroxy-N6-trimethyl-lysine.

(1)H and (13)C nuclear-magnetic-resonance spectroscopy and functional-group analysis were used to determine the molecular structure of an isolated metabolite (II(b)) of trimethyl-lysine as 3-hydroxy-N(6)-trimethyl-lysine, an important intermediate in the conversion of trimethyl-lysine into trimethylammoniobutyrate and carnitine [Hoppel, Cox & Novak (1980) Biochem. J.188, 509-519]. Functional-group analysis revealed the presence of a primary amine and reaction of metabolite (II(b)) with periodate yielded 4-N-trimethylammoniobutyrate as a product, showing 2,3-substitution on the molecule and suggesting that the 3-substitution on the molecule may be an alcohol ([unk]CH-OH), amine ([unk]CH[unk]-NH(2)) or carbonyl ([unk]C=O) functional group. (1)H integration ratios, (1)H and (13)C chemical-shift data and (1)H and (13)C signal multiplicities from the sample (II(b)) were used to complete the identification of metabolite (II(b)) as 3-hydroxy-N(6)-trimethyl-lysine. For example, the proton multiplet at delta 4.2p.p.m. and doublet at delta 4.1p.p.m., positions representative of amine or alcohol substitution on methylene carbon atoms, integration ratios of 1:1:2:9:4 and a positive ninhydrin test suggest 3-hydroxy-N(6)-trimethyl-lysine as the molecular structure for metabolite (II(b)). (13)C chemical-shift data obtained from the sample (II(b)) and compared with several model compounds (trimethylammoniohexanoate, trimethyl-lysine and 3-hydroxylysine) resulted in generation of the spectrum of the metabolite and allowed independent identification of metabolite (II(b)) as 3-hydroxy-N(6)-trimethyl-lysine. The (1)H spectrum of erythro- and threo-3-hydroxylysine are presented for comparison, and the (1)H and (13)C n.m.r. spectra of the erythro-isomer support this analysis.

Carbon Radioisotopes↗
PubMed1981

Rat brain Thy-1 glycoprotein. The amino acid sequence, disulphide bonds and an unusual hydrophobic region.

The full sequence of the Thy-1 membrane glycoprotein of rat brain is reported. The sequence was determined from tryptic and V-8 proteinase peptides and consisted of 111 amino acids. The amino terminus was blocked and consisted of a pyroglutamic acid residue. The molecule contained two disulphide bonds, namely Cys-9--Cys-111 and Cys-19--Cys-85. Three N-linked amino sugars were located at Asn-23, Asn-74 and Asn-98. In each case the sequence on the C-terminal side of the attachment point was Asn-Xaa-Thr as would be expected for N-linkage. The C-terminal peptides were unusual, in that they were either obtained in a highly aggregated form, or could only be purified after binding to Brij 96 micelles. Thus they appeared to have hydrophobic properties, yet did not contain any extended sequence of hydrophobic amino acids. Other unusual features of the C-terminal peptides were the presence of unidentified ninhydrin-positive material and of glucosamine and galactosamine. The C-terminal residue has not been directly identified but Cys-111 is the last conventional amino acid. It is suggested that the hydrophobic properties of the C-terminal peptides may be due to the linkage of lipid. The sequence of the Thy-1 glycoprotein showed homologies with immunoglobulin domains. This relationship is examined in detail in the paper following [Cohen et al. (1981) Biochem. J. 193, 000--000].

Amino Acid Sequence↗
PubMed1981

Interaction between non-classical beta-lactam compounds and the Zn2+-containing G and serine R61 and R39 D-alanyl-D-alanine peptidases.

Streptomyces albus G secretes a Zn2+-containing D-alanyl-D-alanine peptidase. Streptomyces R61 and Actinomadura R39 secrete D-alanyl-D-alanine-cleaving serine peptidases. The effect of non-classical beta-lactam antibiotics on these three model enzymes has been studied. Mecillinam, cefoxitin, quinacillin, quinacillin sulphone, clavulanate and N-formimidoylthienamycin have no effect on the Zn2+-containing enzyme. 6-Amino-penicillanic acid slowly inactivates this enzyme and 7-aminocephalosporanic acid behaves as a reversible inhibitor. Cefoxitin and N-formimidoylthienamycin are potent anti-bacterial agents; they effectively inactivate the serine R39 enzyme and, to a lesser extent, the serine R61 enzyme. All the other beta-lactam compounds tested, including mecillinam, are slow inactivators of these serine enzymes. The intermediates formed between 6-aminopenicillanic acid and the R61 and R39 enzymes are long- and short-lived respectively, whereas those formed between 7-aminocephalosporanic acid and the same R61 and R39 enzymes are short- and long-lived respectively. Breakdown of the short-lived intermediates thus obtained gives rise to several ninhydrin-positive degradation products. The intermediates formed between clavulanate and the serine enzymes are long-lived. With the R39 enzyme, the inactivated complex formed in a first step undergoes subsequent monomolecular rearrangement to give rise to a second species exhibiting a high absorbance at 273 nm.

Actinomycetaceae↗
PubMed1983

Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris).

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.

Animals↗
PubMed1987

The occurrence of N-methyl-D-aspartic acid in muscle extracts of the blood shell, Scapharca broughtonii.

A novel ninhydrin-positive compound, N-methyl-D-aspartic acid, was identified in the muscle extracts of the blood shell, Scapharca broughtonii. This compound is already known to have potent neuroexcitatory activity, inducing hypermotility and strong releasing action of serum luteinizing hormone in mammals. This may be, however, the first finding of N-methyl-D-aspartic acid in natural products.

Animals↗
PubMed1993

A novel ganglioside with a free amino group in bovine brain.

A novel ganglioside which binds cholera-toxin B-subunit was purified from bovine brain by an h.p.l.c. system using an Aquasil column subsequent to Q-Sepharose column chromatography. T.l.c./immunostaining showed that the isolated ganglioside had about 60% of the binding reactivity of the authentic ganglioside GM1 for cholera-toxin B-subunit. On h.p.l.c., this ganglioside migrated between ganglioside GD1a and GD1b, and was found to give positive reactions with ninhydrin and fluorescamine reagents which specifically react with amino groups. The presence of a free amino group was further confirmed by chemical re-N-acetylation. The N-acetylated product had an identical RF value on h.p.l.c. and similar reactivity with cholera-toxin B-subunit as the authentic GM1. H.p.t.l.c., t.l.c./immunostaining, negative-ion fast-atom-bombardment (f.a.b.)-m.s., and 1H-n.m.r. spectroscopy of the novel ganglioside unequivocally demonstrated that it has the basal structure of GM1 with de-N-acetylated neuraminic acid instead of N-acetylneuraminic acid. In the present study we report for the first time that a ganglioside derivative containing de-N-acetylated neuraminic acid, de-N-acetylated GM1, exists in natural brain tissues.

Acetylation↗
PubMed1998

Isolation and characterization of advanced glycation end products derived from the in vitro reaction of ribose and collagen.

An amino acid component, NFC-1, when formed in vitro by the reaction of ribose and protein was shown to comprise a complex mixture of high and low molecular AGE compounds. Two low-molecular-weight components have been successfully isolated and their structure determined. These were alphaNFC-1 [Ndelta-(4-oxo-5-dihydroimidazol-2-yl)-l-ornithine] and betaNFC-1 a 4-imidazolon-2-yl derivative existing in three tautomeric forms. These imidazolone compounds have been shown to originate from the reaction of arginine with glyoxal and methylglyoxal, respectively. A third ninhydrin-positive AGE, gammaNFC-1, was shown to be composed of a number of chromatographically similar compounds which have not yet been characterized.

Animals↗
PubMed1981

Identification of N-monoacetylcystine in uraemic plasma.

1. An unidentified ninhydrin-positive substance of an acidic nature was detected in the plasma of uraemic patients. This substance was isolated from haemodialysate by ion-exchange chromatography and gel filtration, and identified as a sulphur-containing amino acid: N-monoacetylcystine. 2. The quantitative determination of sulphur amino acids in plasma revealed that the plasma levels of cysteic acid, homocysteic acid, taurine, cystine and cystathionine as well as N-monoacetylcystine in uraemic patients were markedly higher than in normal subjects (P less than 0.001 for each). However, the plasma levels of methionine in uraemic patients were within normal limits.

Amino Acids, Sulfur↗
PubMed2002

Characterization and comparison of commercially available German and American cockroach allergen extracts.

BACKGROUND: In this study we examine the variability among unstandardized cockroach allergen extracts. METHODS: We obtained 24 aqueous and glycerinated cockroach allergen extracts from nine manufacturers. We used previously characterized cockroach extracts, E2-Cg and E2-Ca, as references. The modified ninhydrin assay was used to determine protein concentration of each extract. Relative potencies of extracts were determined by competition ELISA, using a human allergic serum pool. Bla g 1 and Bla g 2 levels of glycerinated German cockroach extracts were determined by ELISA using monoclonal antibodies. Extracts were also analysed by SDS-PAGE. RESULTS: Commercial cockroach allergen extracts had highly variable protein contents that were lower than the protein contents of the references. Electrophoretic data confirmed the presence of a variable number and intensity of protein bands in extracts among manufacturers. The relative potencies of the commercial extracts were between 10 and 782 BAU/mL for German cockroach and 10-250 BAU/mL for American cockroach. The mean Bla g 1 content of the commercial extracts was significantly lower than that of the reference (P = 0.001). The mean Bla g 2 content of the commercial extracts was higher than that of the E2-Cg reference but the Bla g 2 levels were more variable compared to Bla g 1. In glycerinated German cockroach extracts, protein concentrations, relative potencies and specific allergen levels were significantly correlated (P < 0.001). CONCLUSION: Our tests indicate that commercially available cockroach allergen extracts are variable in protein content, electrophoretic banding patterns, relative potency and Bla g 2 levels. In glycerinated German cockroach extracts, protein concentrations, relative potencies and specific allergen levels were significantly correlated.

Allergens↗
PubMed2001

Free amino acid concentration in hydatid cyst fluids from fertile and infertile human and animal Echinococcus granulosus.

The aim of this study was to search and compare free amino acid composition of fertile and infertile cyst fluids obtained from humans and animals infected naturally with Echinococcus granulosus, by using automated analysis based on cation-exchange chromatography with post-column ninhydrin derivatization system. 11 free amino acids from fertile (sheep origin), nine from infertile (cattle origin), 13 from infertile (human origin) hydatid cyst fluids and 19 amino acids from sera of patients with hydatid infection were detected. The levels of glycine, alanine, valine and lyrosine in fertile and infertile hydatid cysts fluids were significantly higher than in sera from patients with hydatid cysts. Glycine level in the fertile hydatid cyst fluids (sheep origin) was significantly higher than those of infertile cysts fluids (cattle and human origin) and sera with hydatid patients. Glycine level in fertile hydatid cyst fluids was about two times more concentrated in infertile cattle cyst fluids, 10 times more concentrated in infertile human hydatid cyst fluids and 13 times more concentrated in sera with hydatid patients. On the other hand, alanine and valine concentration in the fertile and infertile cyst fluids were at similar level with the exception that valine level in fertile cyst fluids was 12 times more concentrated in infertile human cyst fluids. The levels of tyrosine, citrulline, leucine, isoleucine and lysine amino acids in fertile and infertile hydatid cyst fluids were similar. Our findings with respect to fertile and infertile cysts fluids showed that free amino acids concentrations in cyst fluids were significantly, higher in sera from patients with hydatid cyst. Total amount of free amino acids content in fertile and infertile cyst fluids was three to eight times higher from that of human sera with hydatid patients.

Amino Acids↗
PubMed1998

Contribution of collateral sprouting to the sensory and sudomotor recovery in the human palm after peripheral nerve injury.

The contribution of collateral sprouting to the sensory and sudomotor recovery was studied in 52 patients aged 3-66 years (mean 35.5 years) from 2 to 9 years following nerve injury and repair. The study included three groups of patients: (1) patients with complete division of median and ulnar nerves (skin reinnervation exclusively due to axon regeneration), (2) patients with isolated division of ulnar or median nerve (skin reinnervation due to axon regeneration and possible collateral sprouting), and (3) patients in whom injured axons failed to regenerate (skin reinnervation exclusively due to collateral sprouting). The end stage of sensory and sudomotor recovery was studied by: clinical methods, sensory nerve action potential (SNAP) measurements, sympathetic skin response (SSR) and the ninhydrin test. We found that recovery of sensory and sudomotor function in groups 1 and 2 was similar. End-stage sudomotor and sensory recovery within the autonomous area of the nerve did not depend on possible collateral reinnervation. Collateral reinnervation from the uninjured nerve was limited to the border innervation area of the palm and ring finger. Adjacent uninjured nerve may contribute to sprouting of nociceptive axons providing a protective function.

Action Potentials↗
PubMed1992

Phenylethylamine and piperidine alkaloids in aloe species.

Of the approximately 300 species of ALOE (Liliaceae) native to Africa and Arabia, leaf extracts of 224 species have been examined chromatographically for alkaloids using ninhydrin, Dragendorff's reagent, nitroprusside, and iodoplatinate as revealing agents. From these, 48 (21%) species contained compounds giving a strong colorimetric reaction with at least one of these reagents. Tyramine derivatives were identified in 18 species and piperidine derivatives in a further 6 species. The other coloured zones remain unidentified but often appeared to represent common amino acids. Some taxonomic correlations are suggested. The presence of the toxic hemlock alkaloids in aloes readily available for potential medicinal use by local inhabitants sounds a note of caution against the unthinking use of these otherwise useful plants.

Journal Article↗
PubMed1998

[Idiopathic autonomic neuropathy. An important disease in differential diagnosis].

HISTORY AND CLINICAL FINDINGS: Two weeks after a mild viral infection a previously healthy 22-year-old woman developed ileus, a severe abnormality of orthostasis, sicca (Sjögren's) syndrome, bilateral miosis and generalized hyphidrosis. Pelvic endoscopy and laparotomy failed to clarify the cause of the mechanical ileus. INVESTIGATIONS: Neurological examination revealed an isolated abnormality of the autonomic system, involving both sympathetic and parasympathetic components. Schellong's, Schirmer's and the ninhydrin tests were markedly abnormal. There was no heart rate variation on breathing and a post-Valsalva hypotensive blood pressure overshoot. Further tests failed to find a cause of the neuropathy. DIAGNOSIS, TREATMENT AND COURSE: The diagnosis of idiopathic panautonomic neuropathy (pandysautonomia) was made. The ileus and hypotension were treated symptomatically with neostigmine, cisapride, midodrine and, initially, with enemas, nasogastric tube feeding and parenteral fluids. The patient was free of symptoms at follow-up examination a year later. CONCLUSIONS: Idiopathic autonomic neuropathy should be considered in the differential diagnosis of functional abnormalities of the sympathetic and/or parasympathetic nervous system, especially in previously healthy young people, in the presence of orthostatic, unexplained gastrointestinal and hidrotic symptoms.

Acute Disease↗