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1.
Porphobilinogen oxygenase, skatole pyrrolooxygenase, and tryptophan pyrrolooxygenase were found in the different parts of germinating wheat (Triticum aestivum) grain seedlings. In the embryos of grains germinated for 24 hours, the activities of PBG oxygenase and skatole pyrrolooxygenase were inhibited by a labile inhibitor. Tryptophan pyrrolooxygenase activity was not inhibited. Embryos of grains germinated for 48 hours showed higher activities for the three enzymes. The latter were also present in the radicles and coleoptiles of 96-hour germinated wheat grains. A DEAE-cellulose analysis of a crude enzymic preparation from embryos allowed the separation of two molecular forms of the three pyrrolooxygenases. The more cationic forms of porphobilinogen oxygenase and skatole pyrrolooxygenase were associated with the inhibitor. This form of porphobilinogen oxygenase had allosteric kinetics while the more anionic form had Michaelis kinetics. Both forms of skatole pyrrolooxygenase had Michaelis kinetics. The activity of tryptophan pyrrolooxygenase was highest in seedling roots and was found to be inhibited in seedling young leaves. This enzyme oxidized tryptophanyl dipeptides, as well as a nonapeptide, to N-formylkynurenine-containing peptides. The pyrrolooxygenase also oxidized the tryptophanyl residues of lysozyme, chymotrypsin, and trypsin.  相似文献   

2.
The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.  相似文献   

3.
Pyrrolooxygenase activities were examined in different varieties of Argentine wheat (Triticum aestivum) which included the traditional Klein varieties and the new mixed Mexican and traditional varieties (DeKalb and Cargill). The enzymatic activities were variety-dependent and were more inhibited in some varieties than in others, while some (Cargill) were devoid of the proteic inhibitor. The enzymes were isolated from the flours as two isoenzymes of different charge whose relative proportions were dependent on the variety of wheat used. The more cationic isoenzymes were eluted with 10 mM Tris-HCl buffer (pH 7.6 from DEAE-cellulose and the less cationic were eluted with 50 mM NACl in the same buffer. The protein inhibitor, when present, was associated with the more cationic isoenzymes. Porphobilinogen oxygenase and skatole pyrrolooxygenase activities were higher in the endosperm, while tryptophan pyrrolooxygenase activity was higher in the embryo. The proteic inhibitors were mainly concentrated in the embryo.  相似文献   

4.
Both porphobilinogen oxygenase and skatole pyrrolooxygenase of wheat germ have isoenzyme forms of different charge. The more cationic isoenzymes were eluted from DEAE-cellulose with 10 mM Tris-HCl buffer (pH 7.6) and the less cationic were eluted with 50 mM NACl in the same buffer. The former had almost twice as many free amino groups (per mg of protein) as the latter. The more cationic isoenzyme was more sensitive to chelating agents and to acid treatment. They were differently inhibited by sodium dodecyl treatment and by temperature inactivation. Porphobilinogen oxygenase isoenzymes showed different activities with different buffers and also differed in their kinetics.  相似文献   

5.
1. Isolation conditions for rainbow trout (Salmo gairdneri) liver ribosomes were optimized. 2. Optimal initial buffer (Buffer I) concentrations were 250 mM sucrose, 50 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 5 mM MgSO4.7H2O. 3. Optimal concentrations for post-105 supernatant buffer (Buffer III) were 25 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 8 mM MgSO4.7H2O.  相似文献   

6.
Leaf extracts of pepper (Capsicum annuum) and poinsettia (Euphorbia pulcherrima) contained pyrrolooxygenases which varied in activity according to the age of the leaves and the origin and physiological condition of the plants. An inhibition of the pyrrolooxygenases was present in the crude extracts of senescent leaves. Fruiting enhanced pyrrolooxygenase activity and added a new ionic form of greater negative charge to the usual cationic form of the enzymes. Pyrrolooxygenases of C. annuum leaves from greenhouse-grown plants showed three forms of different ionic charge which exhibited multiple MW forms for porphobilinogen oxygenase and skatole pyrrolooxygenase. The cationic form of porphobilinogen oxygenase had sigmoidal kinetics, while the anionic forms had Michaelis kinetics. Skatole and tryptophan pyrrolooxygenase showed Michaelis kinetics. Pyrrolooxygenase activities in E. pulcherrima were lower than those in C. annuum and the former were also found to be more unstable.  相似文献   

7.
Summary A new type of oxygenases was isolated from plant and animal sources which oxidized pyrrole and indole derivatives. They had a broad substrate specificity and were called pyrrolooxygenases. Three different enzymes within the group were identified; skatole pyrrolooxygenase, tryptophan pyrrolooxygenase and porphobilinogen oxygenase. The first two oxidized the pyrrole ring of the various indole derivatives affording substituted o-formanidophenacyl derivatives as the main oxidation products. Tryptophan pyrrolooxygenase also oxidized the tryptophanyl residues of peptides and enzymes. When those residues were essential for the activity of the tryptophan containing enzymes, then inactive enzymes were obtained.Porphobilinogen oxygenase oxidized porphobilinogen and related alkylpyrrole compounds affording 3-pyrrolin-2-one derivatives. The pyrrolooxygenases acted as mixed-function oxidases, since they required the presence of oxygen and of a reducing agent. The substrate, the oxygen and the reductant were consumed in equimolar amounts. The best artificial reducing agent was sodium dithionite. Illuminated active chloroplasts were the natural reducing agent of the plant enzymes and NADPH was the reducing agent of the animal enzymes. Pyrrolooxygenases were located in the chloroplasts of green leaves and in the microsomes in the case of the mammalian enzymes. The activity of the enzymes in the crude extracts was usually low, due to the presence in the same of a protein inhibitor. When the inhibitor was separated by protein fractionation methods, full enzymatic activity was recovered. Destruction of the inhibitor by aging or by temperature had the same effect. The very low oxygenase activity present in the microsomal rat liver preparations could be strongly enhanced by previous administration to the rats of phenobarbital or steroids. This induction of the oxygenase activity was coincident with a drop in the amount of inhibitor present in the extracts.The properties and metabolic role of the pyrrolooxygenases are discussed.  相似文献   

8.
9.
Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.  相似文献   

10.
11.
Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

12.
Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.  相似文献   

13.
R O Ryan  L D Hicks  C M Kay 《FEBS letters》1990,267(2):305-310
Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0/100 mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KCl concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent S20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent S20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KCl which yielded a particle Mr = 887,000. Circular dichroism spectra of monomeric LTP sample revealed 6% alpha-helix, 49% beta-sheet, 7% beta-turn and 35% random coil while aggregated LTP contained 13% alpha-helix, 66% beta-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.  相似文献   

14.
The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) n .  相似文献   

15.
The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) n .  相似文献   

16.
Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.  相似文献   

17.
The affinity of eukaryotic tyrosyl-tRNA synthetases from bovine liver and from yeast for E. coli ribosomal RNA and synthetic polyribonucleotides has been studied by protein binding on the rRNA-Sepharose column and enzyme inhibition by high molecular weight RNAs. Tyrosyl-tRNA synthetase from bovine liver (Mr 2.59 kDa) was fully retained on the rRNA-Sepharose and eluted by buffer with 100 mM KCl. The functionally active modified form of bovine liver tyrosyl-tRNA synthetase obtained by endogenous limited proteolysis (Mr 2.38 kDa) partially maintains the affinity for rRNA and is eluted by 50 mM KCl. The highest rRNA-binding ability was revealed for yeast tyrosyl-tRNA synthetase eluted by 200 mM KCl. The E. coli tyrosyl-tRNA synthetase was not retained on rRNA-Sepharose. The aminoacylation activities of both bovine liver and yeast tyrosyl-tRNA synthetases were efficiently inhibited by rRNA and the inhibition was partially competitive in respect to tRNA(Tyr). At the same time the activities of proteolytically modified bovine tyrosyl-tRNA synthetase and E. coli tyrosyl-tRNA synthetase were not influenced by the addition of rRNA. Synthetic single- and double-stranded polyribonucleotides specifically inhibited the activity of bovine tyrosyl-tRNA synthetase to different extent. The inhibition degree of bovine liver tyrosyl-tRNA synthetase decreased in the order: poly (G) greater than poly (I) greater than poly (I).poly (C) greater than poly (G).poly (C) greater than poly (C) greater than poly (A). Poly (U) did not inhibit the activity of bovine liver tyrosyl-tRNA synthetase.  相似文献   

18.
Phosphoenolpyruvate carboxylase (PEPC) EC 4.1.1.31 was extracted from nodules and roots of 2-day-old seedlings of lupin (Lupinus luteus L.). Chromatography on DEAE-cellulose of the nodule extract gave two forms of the enzyme: PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, respectively. A third form PEPC III from lupin roots was eluted from DEAE-cellulose column at the same buffer concentration as PEPC II from nodules. PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, more active in the 6-week-old nodules binding effectively nitrogen than in the 12-week-old ones.  相似文献   

19.
Chemical modifications of rye seed chitinase-c (RSC-c) with various reagents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity. Of these, the modification of tryptophan residues with N-bromosuccinimide (NBS) was investigated in detail.

In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the activity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72.

The NBS-oxidation of Trp72 at pH 5.9 was protected by a tetramer of N-acetylglucosamine (NAG4), a very slowly reactive substrate for RSC-c, and the activity was almost fully retained. In the presence of NAG4, RSC-c exhibited an UV -difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and involved in the binding to substrate.  相似文献   

20.
Wang H  Ng TB  Liu Q 《Life sciences》2002,70(8):877-885
From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin.  相似文献   

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