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)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C. It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+. When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+. A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin. This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means. For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively. The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis. A Ki value of 10.5 mM for NADP+ was derived from inhibition studies. The principal conclusion from these studies is that NAD+ binding to L. mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding. Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzyme's conformation, as reflected in a much higher KD value. This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.
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PMID:Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: ligand-induced conformational changes. 329 33

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.
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PMID:Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii. 335 5

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.
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PMID:Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides. 337 61

A strong inhibitor of G6PDH has been detected in rat liver homogenates. The inhibitor, isolated by ultrafiltration methods, proved to be very stable under incubation with trypsin and high temperatures. Gel-filtration through Sephadex G-75 showed it to have a molecular weight of 3,500 daltons, though perchloric acid treatment produced a light form of 900 daltons. Both forms of inhibitor act as competitive inhibitor with respect to G6P and exhibit non-competitive inhibition pattern with respect to NADP+. Physical and kinetic properties, and the increase of G6PDH activity at low NADP+ concentrations, in the presence of NADPH and inhibitor or palmitoyl-CoA, in relation to the G6PDH activity in presence of NADPH, lead to the identification of the low-molecular weight inhibitor as palmitoyl-CoA.
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PMID:[Characterization of an inhibitor of glucose-6-phosphate dehydrogenase]. 361 14

Purified pea chloroplast malate dehydrogenase (NADP) was reduced, S-pyridylethylated with 4-vinyl-pyridine and cleaved with trypsin. The resulting peptides were separated by reversed-phase high-performance liquid chromatography. Several of these peptides were subjected to automated Edman degradation. The sequences obtained were compared to the published primary structures of malate dehydrogenase from the thermophilic bacterium Thermus flavus and with the sequence of heart mitochondrial and cytoplasmic malate dehydrogenase (NAD). Most peptides from choroplast malate dehydrogenase (NADP) showed high homology with sequences of the other malate dehydrogenases, especially with those of the bacterial enzyme. One of the sequenced peptides contains the active-site histidine residue which is conserved in all malate dehydrogenases. Our results suggest a common evolutionary origin for all malate dehydrogenases despite their different coenzyme specificities and regulatory properties. The sequenced peptides which revealed no homology were either located at the amino-terminal or at the carboxy-terminal region of chloroplast malate dehydrogenase (NADP). These novel sequences are most likely plant-specific extensions of an ancestral malate dehydrogenase and may be responsible for the unique light-dependent activation of the chloroplast enzyme.
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PMID:Amino acid sequence similarity between malate dehydrogenases (NAD) and pea chloroplast malate dehydrogenase (NADP). 366 38

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.
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PMID:Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers. 395 68

Spinach ferredoxin-NADP+ oxidoreductase was inactivated by treatment with 2',3'-dialdehyde NADP+ (periodate-oxidized NADP+), which selectively modifies a lysine residue at the nucleotide-binding domain of the enzyme. The identity of the derivatized residue was ascertained by thin-layer chromatography of the protein hydrolysate. Reductase that had been labeled with periodate-oxidized NADP+ and NaB3H4 was treated with trypsin, and samples of the tryptic digest were subjected to reverse-phase high-performance liquid chromatography. The radioactivity profiles showed modification of one specific peptide. The primary structure of this peptide was found to be Gly-Glu-Lys*-Met-Tyr-Ile-Gln-Thr-Arg, where Lys* represents the derivatized lysine. The sequence obtained corresponds to residues 242-250 in the primary structure of spinach ferredoxin-NADP+ reductase recently reported [Karplus et al. (1984) Biochemistry 23, 6576-6583].
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PMID:Isolation and sequencing of an active-site peptide from spinach ferredoxin-NADP+ oxidoreductase after affinity labeling with periodate-oxidized NADP+. 401 97

After intracardial injection of [1,2-3H]dehydroepiandrosterone ([3H]DHA) into female rats, [3H]DHA was found to accumulate and was metabolized in the preputial gland, but not in the diaphragm. The identified metabolites of [3H]DHA in the preputial gland were delta 4-androstenedione-3 alpha, 17 beta-diol. Cells were isolated from the preputial gland after treatment with trypsin and collagenase III, and centrifugation in Ficoll gradients. Activity of the enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase delta 4-5-isomerase (3 beta-HSD) responsible for transforming DHA into delta 4-androstenedione was found mainly in the 105,000 g pellet (microsomal fraction) of homogenates of the isolated cells. It used preferentially NAD over NADP as a coenzyme, with a pH optimum at 8.5. The apparent Km for DHA was 5.5 X 10(-5) M, and the Vmax was 1.72 nmol/min/mg microsomal protein. These findings indicate that DHA is preferentially taken up by the preputial gland where it undergoes metabolism to form more potent androgens, and suggest that DHA may have important androgenic influence on the preputial gland.
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PMID:delta 5-3 beta-Hydroxysteroid dehydrogenase delta 4-5-isomerase activity and metabolism of dehydroepiandrosterone in rat preputial gland. 623 67

Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.
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PMID:Formation of an iso-1-cytochrome c-like species containing a covalently bonded heme group from the apoprotein by a yeast cell-free system in the presence of hemin. 624 50

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.
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PMID:Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells. 633 18


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