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)

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparations, but with a small shift to the blue of the alpha-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.
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PMID:Properties and reconstitution of a cytochrome oxidase deficient in subunit III. 630 85

A trypsin-degradable nerve growth factor (NGF) receptor associated with the phospholipid component of the surface membrane has been detected on F98 anaplastic glioma cells. NGF also bound to the nucleus of F98 cells. Bound NGF was not displaceable by insulin, cytochrome C, growth hormone, or bovine serum albumin. Specific binding of NGF occurred with a Kd of 8.79 X 10(-12) M as determined by Scatchard analysis with approximately 34,000 receptors per cell. Specific NGF binding was also evident to C6 rat glioma cells and IMR-32 human neuroblastoma cells, but not to 3T3 mouse fibroblasts. These observations coupled with previous findings suggest that the NGF receptor may be a marker found on cells of neural derivation. As little as 1 ng/ml NGF caused an increase in the adhesiveness of F98 cells to culture flasks. Increased adhesiveness could be observed in as little as 5 min and was apparent for at least 45 min. At 25 min in NGF-containing medium, 24 +/- 3% of the cells adhered to the flasks compared to 13 +/- 1% of control cells. The NGF-induced increase in adhesiveness was not duplicated by epidermal growth factor, insulin, cytochrome c, bovine serum albumin, dibutyryl cyclic AMP, or sodium butyrate. Oxidized NGF blocked the effect of native NGF, but had little or no adhesion-promoting activity itself. Pretreatment of the cells with NGF was also effective in promoting adhesion, even though nerve growth factor was not added to the binding medium. The effect of this pretreatment was reversible; when NGF-pretreated cells were grown in medium without supplemental NGF, the adhesiveness of the cells returned to control levels or lower.
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PMID:Increased adhesion response of anaplastic glioma cells to nerve growth factor and the presence of specific receptors. 631 24

A method for purification of detergent-solubilized cytochrome b5 to gel electrophoretic homogeneity from yeast (Saccharomyces cerevisiae) microsomes is described. The purified preparation shows the same absorption spectra as the trypsin-solubilized cytochrome (Y. Yoshida, H. Kumaoka, and R. Sato J. Biochem. 75, 1211-1219 (1974)) in the visible and Soret regions. The detergent-solubilized cytochrome is an amphipathic protein having a monomeric molecular weight of about 18,000 and exists as a hexa- or heptameric aggregate (Mr 122,000) in aqueous media. In the presence of low concentrations of Triton X-100, it interacts effectively with the intact form of NADH-cytochrome b5 reductase purified either from yeast microsomes or from rabbit liver microsomes. Upon trypsin digestion, it is converted to a heme-containing, hydrophilic fragment (Mr 13,000) which retains the spectral characteristics of the original cytochrome, does not form aggregates, and interacts with the reductase only poorly. It is concluded that the preparation purified in this study represents the intact form of yeast cytochrome b5 consisting of a hydrophilic, heme-containing moiety (Mr 13,000) and a hydrophobic, membrane-binding tail (Mr 5000).
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PMID:Purification and characterization of intact cytochrome b5 from yeast microsomes. 633 56

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.
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PMID:Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides. 636 48

Two forms of cytochrome P-450 have been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated with imidazole. Several criteria indicate that the cytochrome of higher electrophoretic mobility is identical with ethanol-inducible isozyme 3a. "Imidazole-3a" and "ethanol-3a" exhibit the same chromatographic characteristics and have identical electrophoretic mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the two protein preparations have the same absorbance maxima and absorption coefficients in the oxidized, reduced, and reduced-CO states. A single immunoprecipitin band exhibiting complete identity was observed upon reaction of imidazole-3a and ethanol-3a with the immunoglobulin G fraction from sheep immunized with the latter protein. The amino acid composition and first 10 residues of the amino terminus of the two protein preparations are indistinguishable, as are the high-performance liquid chromatographic maps of the peptides obtained upon cleavage with trypsin, Staphylococcus aureus V8 protease, or Lys C endoproteinase . Furthermore, these preparations have very similar activities in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline. Evidence was obtained that the cytochrome of lower electrophoretic mobility isolated from imidazole-treated rabbits is probably identical with isozyme 6; the spectral characteristics, amino acid composition, and carboxyl-terminal sequence are described. As an inducer, imidazole has the advantage over ethanol of being less variable in its effects and requiring a shorter period of treatment. From the resulting liver microsomes, one can readily isolate, in addition to P-450 isozymes 3a and 6, isozymes 3c and 4 as well as epoxide hydrolase.
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PMID:Purification of liver microsomal cytochrome P-450 isozymes 3a and 6 from imidazole-treated rabbits. Evidence for the identity of isozyme 3a with the form obtained by ethanol treatment. 642 1

Methods are described for incorporation of purified forms of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450LM2, P-450LM3 and P-450LM4 (LM, liver microsomes) into phospholipid vesicles. It was found that each cytochrome could individually be incorporated into preformed phospholipid vesicles in the absence of cholate. However, NADPH-cytochrome P-450 reductase prevented incorporation of P-450 by this method, a phenomenon possibly inherent in the formation of complexes between P-450 and the reductase in solution. Using the cholate-gel filtration technique it was possible to prepare monolamellar phosphatidylcholine vesicles containing any of the cytochromes and P-450 reductase in good yields. It was found that P-450LM3-containing vesicles had a mean diameter of 47 nm, whereas vesicles formed under the same conditions but containing P-450LM4 were much smaller (mean diameter 33 nm). Vesicles formed with P-450LM2 were homogeneous in density (1.04 g/cm3) according to isopycnic centrifugation in Ficoll but not in size (44-72 nm). These findings, taken together with results obtained from treatment of the cytochromes in soluble form and in reconstituted vesicles with the non-penetrating reagent, p-diazobenzene sulphonate, indicate a unidirectional, relatively peripheral orientation of P-450LM4 with the major part localized on the outside of the vesicles. Experiments with trypsin and cytochrome c-reduction demonstrated a unidirectional orientation of P-450 reductase towards the outside of the vesicles.
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PMID:Incorporation of purified components of the rabbit liver microsomal hydroxylase system into phospholipid vesicles. 677 67

Chromatography of electrophoretically homogeneous cytochrome P-450LM4 from cholestyramine-treated rabbits on octylamine-Sepharose resulted in the isolation of two subfractions, cytochrome P-450LM4 I and cytochrome P-450LM4 II, with different catalytic properties. The original cytochrome P-450LM4 fraction catalyzed 7 alpha-hydroxylation of cholesterol, 12 alpha-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 6 beta-hydroxylation of testosterone, and demethylation of ethylmorphine. Cytochrome P-450LM4 I was inactive in cholesterol 7 alpha-hydroxylation, but catalyzed the other hydroxylations. Cytochrome P-450LM4 II catalyzed efficient cholesterol 7 alpha-hydroxylation. It also catalyzed the other hydroxylations, although at lower rates than cytochrome P-450LM4 I. Emulgen inhibited all steroid hydroxylase activities in cytochrome P-450LM4 II except the cholesterol 7 alpha-hydroxylase activity. Cytochrome P-450LM4 I and cytochrome P-450LM4 II showed the same apparent molecular weight and spectral properties as the original cytochrome P-450LM4 fraction. The two subfractions differed in amino acid composition. They produced similar but not identical one-dimensional peptide maps upon limited proteolysis with papain, chymotrypsin, and trypsin. The results show that cytochrome P-450LM4 from cholestyramine-treated rabbits contains at least two species with different amino acid compositions and different substrate specificities toward C27-steroids involved in biosynthesis of bile acids.
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PMID:Hydroxylations in biosynthesis of bile acids. Isolation of subfractions with different substrate specificity from cytochrome P-450LM4. 681 87

A method for biomembrane reconstitution from microsomal proteins and lipids solubilized by sodium cholate consisting in a removal of the detergent by its dialysis followed by treatment with 10% albumin has been developed. A comparison of the original and reconstituted membranes showed that the phospholipid, protein and enzymatic composition of the latter is similar or only slightly different from that of the original ghosts. The reconstituted membranes contained 1.5 times more cytochrome b5 and an equal amount of cytochrome P-450. No more than 20% of cytochrome P-450 was represented by the inactive form. The inactivation rate of the reduced hemoprotein in the reconstituted membranes was lower than in the ghosts. Both in the reconstituted and original membranes the similarity of solubilization patterns of microsomal electron carries upon trypsin treatment was indicative of identical topography of these proteins. The most effective was the reconstitution of NADH and cumole hydroperoxide-dependent N-demethylase, whereas the p-hydroxylase and O-dealkylase activities of the reactivated P-450 were not retained. Hence, no complete reconstitution of the properties of the microsomal membrane and its redox chain was observed in spite of the effective removal of the detergent, similar localization of microsomal electron carriers in the reconstituted membranes and ghosts and the reconversion of cytochrome P-420 into cytochrome P-450.
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PMID:[Comparative study of original and reconstituted by self-assembly endoplasmic reticulum membranes]. 729 23

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.
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PMID:Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420. 740 43

Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4.
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PMID:A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains. 743 81


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