Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regression and turnover of the surface glycoconjugates of trypsin-prepared pig and human cultured epidermal cells have been determined using the glycoprotein precursors N-acetyl-D-(I-3H) glucosamine (3H-NAG) and N-(3H)-acetyl-D-mannosamine (3H-NAM). Sialic acid assays have been performed on similar unlabelled cells. The major points which emerged from this study were: (1) Trypsin-damaged cell surfaces are rapidly repaired, probably by normal membrane turnover. There was a 12% regeneration of sialic acid within 2 h and total resynthesis occurred within 24 h. (2) The presence of an internal membrane system, part of which also demonstrates turnover, probably contributed to the speed of surface membrane repair. Some of the glycoprotein/sialic acid of this internal membrane system (30%) remains bound for a considerable length of time. (3) The membrane turnover maintains the cell in equilibrium so that total loss equals the synthesis of glycoprotein. (4) The equilibration of 3H-NAG or 3H-NAM uptake between 24 and 48 h is limited by the relative concentrations of glucose and labelled sugar in the medium at this time. (5) 3H-NAm was a more specific marker of glycoprotein than 2H-NAG. (6) The results for human epidermal cells closely matched those for pig epidermal cells, indicating that pig cells can be used as a model for human cells.
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PMID:Synthesis and turnover of membrane glycoconjugates in monolayer culture of pig and human epidermal cells. 724 76

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5'-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPH via NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.
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PMID:A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation. 776 Jun 89

Many previous reports using experimental animal models of pancreatitis have suggested that oxygen free radicals play an important part in initiation and development of pancreatitis. Infiltration of inflammatory cells--that is, neutrophils, lymphocytes, and monocytes--has been seen in damaged pancreatic glands of animal models and patients with pancreatitis. As neutrophils are known to be the highest producer of oxygen free radicals among these inflammatory cells, it seems plausible that oxygen free radicals produced by neutrophils have some pathoaetiological meaning in pancreatitis. This study measured the superoxide production by neutrophils obtained from patients with acute and chronic pancreatitis and then examined the effects of pancreatic enzymes on superoxide production. Patients showed significantly higher superoxide production by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) stimulated neutrophils than healthy controls. Among the three pancreatic enzymes, amylase, trypsin, and elastase, elastase was the only one that increased the superoxide production by PMA stimulated neutrophils, by an increment of 1.5-fold. It also increased the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase prepared from PMA stimulated neutrophils by a factor of 2.1. High affinity and low affinity binding sites for elastase on neutrophils were identified. These results suggest that elastase plays a part in the development of pancreatitis by enhancing superoxide production of neutrophils.
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PMID:Specific interaction of pancreatic elastase and leucocytes to produce oxygen radicals and its implication in pancreatitis. 782 93

Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [alpha-32P]nicotinamide-2-azidoadenine dinucleotide has been used to identify an NADH/NADPH binding site. Specificity of nucleotide interaction was demonstrated by saturation and protection of labeling at physiologically relevant concentrations. Saturation of photoinsertion was observed at approximately 100 microM probe with an apparent Kd of approximately 25 microM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 microM of the above-mentioned compounds. The protection obtained with NADP+ and NADPH was of the same order, respectively. The adenine ring binding domain of NADH/NADPH binding site was identified by trypsin and chymotrypsin digestion of the photolabeled prolactin and purification of the photolabeled peptide by boronate affinity chromatography and immobilized Fe3+ affinity chromatography. The peptide was identified to be Ala22-Tyr28. These studies demonstrate that prolactin contains an NADH/NADPH binding site which may be significant in the mechanism of action of this hormone.
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PMID:Identification and characterization of a nucleotide binding site of ovine prolactin with 2-azido-NAD. 832 98

We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk-signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.
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PMID:A chromaffin cell-derived protein induces the NADPH-diaphorase phenotype in cultured rat spinal cord neurons. 868 18

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.
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PMID:Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia. 887 22

1. Cell-free excised membrane patches were used to examine the properties of a novel nicotinamide-adenine dinucleotide (beta-NAD+)-activated ion channel in the rat insulin-secreting cell line, CRI-G1. 2. In inside-out recordings, beta-NAD+ (0.05-1.0 mM) induced the appearance of a channel characterized by extremely slow kinetics, with mean open times in the range of seconds. The estimated EC50 for activation was 114 microM. Channel activity declined with time (run-down) following activation by beta-NAD+ in excised patches and this was not prevented by intracellular application of trypsin. 3. The single channel current-voltage relationship was linear with a conductance of 74 pS in symmetrical NaCl. The channel appears equally permeable to Na+, K+ and Cs+, exhibits an appreciable permeability to Ca2+, Mg2+ and Ba2+, but excludes anions. 4. The channel displays an unusual voltage sensitivity, with an abrupt increase in open-state probability at depolarized voltages. 5. Channel opening, in the presence of beta-NAD+, required both Ca2+ and Mg2+ to be present at the internal side of the membrane. Activation by Ca2+ required a concentration of at least 10 microM and was maximal at 0.1 mM. Ba2+ did not substitute for Ca2+ in inducing channel activity nor did it inhibit activation by Ca2+. Increasing the concentration of intracellular Mg2+ stabilized the open state of NAD(+)-activated channels. 6. The non-selective cation channel reported here differs in its gating and modulatory characteristics from non-selective cation channels described in other tissues. This channel may play a role in the pathophysiological responses of beta-cells to oxidative stress.
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PMID:Characterization of a nicotinamide-adenine dinucleotide-dependent cation channel in the CRI-G1 rat insulinoma cell line. 940 72

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The X indicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+ binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.
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PMID:Identification of an NAD+ binding site of brain glutamate dehydrogenase isoproteins by photoaffinity labeling. 981 15

Recessive congenital methemoglobinemia due to nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 clinical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive congenital methemoglobinemia, the patients from which were diagnosed according to clinical symptoms and b5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolated total RNA from the peripheral leukocytes of the propositus and b5R complementary DNA (cDNA) by reverse transcription- polymerase chain reaction (RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing the cloned PCR products. The results showed that the propositus was homozygous for a G-->A transition at codon 203 in exon 7, changing a cysteine to a tyrosine (Cys203Tyr). To characterize the mutant enzyme, both glutathione S-transferase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expressed in Escherichia coli and affinity purified. The results showed that the catalytic activity of the enzyme was not much affected by this amino acid substitution, but the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin. These properties of the mutant enzyme would account for the restricted b5R deficiency and mild clinical manifestations of these type 1 patients. The finding of this novel mutation makes codon 203 the only position within the b5R gene at which more than 1 mutation has been found.
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PMID:A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia. 1080 96

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site. Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site. The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants. The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs. The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+). In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.
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PMID:Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase. 1219 7


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