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

---

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

In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.
...
PMID:Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. 310 60

Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.
...
PMID:NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110. 313 23

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.
...
PMID:Limited proteolysis of the nitrate reductase from spinach leaves. 319 46

Cytochromes P-450 LM2 and P-450 LM4 from rabbit liver microsomes were chemically modified with tetranitromethane. Nitration of two tyrosine residues of both isozymes inhibits the benzphetamine N-demethylase activity of P-450 LM2 as well as the p-nitrophenetole O-deethylase activity of P-450 LM4 by about 80%. For identification of the modified tyrosine residues the inactivated enzymes were digested with trypsin, and the labeled peptides were separated by HPLC. Sequencing of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM2 showed that the tyrosine residues at positions 235 and 380 were nearly fully nitrated, while Tyr-348, Tyr-484 and Tyr-111 were only partially labeled (about 40-50%). In the presence of the heme-binding inhibitor metyrapone, Tyr-380 is partially protected against modification, and the extent of inactivation is diminished as well. These results suggest that Tyr-380 of cytochrome P-450 LM2 presents a catalytically essential amino acid residue at its active center. Sequence analyses of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM4 revealed that mainly Tyr-243 and Tyr-271 were labeled, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent (about 30-45%). Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are suggested to be functionally involved in the interaction with NADPH-cytochrome P-450-reductase.
...
PMID:Comparative studies on the accessibility and functional importance of tyrosine residues in cytochrome P-450 isozymes. 320 47

An enzyme preparation (IIIB) isolated from liver microsomes of untreated male rats was found to contain two activities--short-chain trans-2-enoyl-CoA hydratase and beta-ketoacyl-CoA reductase. The hydratase was purified more than 1000-fold, while the reductase activity was purified over 600-fold. Employing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a single band with a molecular weight of 76,000 was observed. Although attempts to separate these two activities have failed, it remains to be established whether the final preparation contains a single enzyme with two activities or two separate enzymes. The hydratase was most active toward crotonyl-CoA, followed by trans-2-hexenoyl-CoA (6:1) and -octenoyl-CoA (8:1); the enzyme was essentially inactive toward substrates containing more than eight carbon atoms. The Vmax for crotonyl-CoA was 2117 mumol/min/mg protein, while the Km was 59 microM. Using acetoacetyl-CoA as substrate, the Vmax for the beta-ketoacyl-CoA reductase was over 60 mumol/min/mg protein and the Km was 37 microM; the Vmax for beta-ketopalmitoyl-CoA was only 15% of that observed with acetoacetyl-CoA, although the Km was 6 microM. During the course of purification, a second short-chain hydratase was discovered (fraction IVA); unlike IIIB, this fraction catalyzed the hydration of 4:1, 6:1, and 8:1 at similar rates. The partially purified preparation yielded maximal activity with 8:1 CoA (apparent Vmax 35 mumol/min/mg), followed by 6:1 CoA, 4:1 CoA, and 10:1 CoA; longer chain CoA's were relatively poor substrates, with trans-2-hexadecenoyl CoA about 0.1 as active as 8:1 CoA. On SDS-gels, fraction IVA contained four bands, all of which were below 60,000 Mr. Proteases, such as trypsin, chymotrypsin, and subtilisin, were found to completely inactivate both enzyme fractions.
...
PMID:Isolation of rat liver microsomal short-chain beta-ketoacyl-coenzyme A reductase and trans-2-enoyl-coenzyme A hydratase: evidence for more than one hydratase. 351 72

The activities, properties, and steady-state kinetics of the five enzymes catalyzing the synthesis of 1-acyl- and 1-alkyl-sn-glycerol 3-phosphate in the cultured skin fibroblasts from Zellweger syndrome patients and normal controls were studied in detail. Judging from their Km and Vmax values, glycerol phosphate acyltransferase (EC 2.3.1.15), acyl/alkyl dihydroxyacetone phosphate reductase (EC 1.1.1.101), and acyl coenzyme A reductase (long-chain alcohol forming), appear to be affected only slightly by the absence of peroxisomes characteristic of the Zellweger syndrome. Glycerophosphate acyltransferase also showed no differences in N-ethylmaleimide sensitivity nor in inhibition by dihydroxyacetone phosphate between these cell types. Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) and alkyl dihydroxyacetone phosphate synthase (EC 2.5.1.26) have altered activity and kinetic constants in homogenates from Zellweger syndrome fibroblasts. Dihydroxyacetone phosphate acyltransferase has similar Km (DHAP) values in both control and Zellweger syndrome cells; however, the value for the Vmax in Zellweger syndrome cells is only 6% of that found in the controls. This is interpreted as indicating that this enzyme is not defective in this disease but is simply present at a depressed level. Also, this enzyme activity has a maximum rate at pH 7.0-7.5 in the mutant cells as opposed to pH 5.4 in the controls. Acylation of dihydroxyacetone phosphate by control cell homogenate was stimulated by N-ethylmaleimide at both pH 5.7 and 7.5 whereas this activity from Zellweger syndrome cells was slightly inhibited at pH 5.7 and strongly inhibited at pH 7.5. In the absence of detergent, dihydroxyacetone phosphate acyltransferase in the Zellweger syndrome cells was much more labile to trypsin than in the control cells. Alkyl dihydroxyacetone phosphate synthase had a slightly higher Km (33 vs 17 microM) for palmitoyl dihydroxyacetone phosphate and a lower Vmax (0.07 vs 0.24 mU/mg protein) in the Zellweger syndrome cells as compared to controls. Although this is a substantial decrease in activity, it probably contributes little to the decreased rate of ether lipid synthesis in these cells. The major problem in this respect is apparently the loss of dihydroxyacetone phosphate acyltransferase activity. All of these enzymes, in both control and Zellweger syndrome cell homogenates, are sedimentable by centrifugation at 100,000g. Also, with the exception of dihydroxyacetone phosphate acyltransferase they had similar patterns of inactivation by heat in both cell types.
...
PMID:Properties of the enzymes catalyzing the biosynthesis of lysophosphatidate and its ether analog in cultured fibroblasts from Zellweger syndrome patients and normal controls. 364 70

Solubilization by sodium deoxycholate and trypsin of some metabolic enzymes of unrelated compounds associated with endoplasmic reticulum membranes was carried out. The effects of urea, butanol and detergents on the retinol content in the membranes were studied. It was shown that retinol deficiency causes changes in the interactions of NADH-arylesterase with microsomal membrane components that are manifested in the decrease of the activating effect of butanol and low detergent concentrations on the NADH-reductase activity as well as in the increase in the damaging effect of urea and high detergent concentrations on the enzyme activity. Under conditions of retinol deficiency, the degree of solubilization of NADH-reductase, hydroxylase and arylesterase in the presence of sodium deoxycholate is enhanced. After treatment of liver microsomes of retinol-deficient animals with trypsin or with a trypsin-sodium cholate mixture, the content of these enzymes in the supernatant becomes much greater than that in liver microsomes of vitamin A-deficient rats. It is assumed that retinol deficiency causes of weakening of hydrophobic interactions within the membrane as well as partial translocation of the enzymes from the hydrophobic to the hydrophilic layer.
...
PMID:[Effect of retinol deficiency on the activity and solubility of various enzymes of the endoplasmic reticulum membranes in the rat liver]. 369 12

The membrane domain of NADH-cytochrome-b5 reductase, which extends from the amino-terminal myristic acid through the first 28 amino acid residues, can be isolated in cholate after a mild trypsin treatment of cholate-solubilized reductase, and in phospholipid vesicles after exhaustive trypsin treatment of vesicle-bound reductase. The detergent-solubilized peptide has a high affinity for phospholipid vesicles and can be reconstituted in vesicles by the detergent-dialysis method. The fluorescence of Trp-16 of this peptide is highly sensitive to the polarity of the microenvironment. The fluorescence quantum yield of this residue is 0.10 when the peptide is dispersed in 1% sodium cholate, but 0.46-0.52 when the peptide is reconstituted in phospholipid vesicles. Fluorescence energy transfer from this tryptophan residue in vesicle-bound peptide to a random array of acceptors in the head-group region of the vesicle outer monolayer shows that Trp-16 resides at a depth of 20-23 A in the bilayer.
...
PMID:Binding and fluorescence properties of the membrane domain of NADH-cytochrome-b5 reductase. Determination of the depth of Trp-16 in the bilayer. 371 Oct 89

The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Subcellular distribution of leukotriene C4 binding units in cultured bovine aortic endothelial cells. 374 19

[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 microM and 15 microM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed--16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.
...
PMID:Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway? 377 60


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---