UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
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The H-D exchange of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) in D2O was studied by an ultraviolet spectroscopic method recently proposed (J. J. Englander, D. B. Calhoun, and S. W. Englander, (1979) Analytical Biochemistry, 92, 517-524). Isotopic exchange data are presented as plots of X (the fraction of unexchanged peptide hydrogen atoms at time t) versus log(kot), where ko is the pH dependent rate constant for peptide groups exposed to the solvent. In the range of pD 2.25-6.9, at 20 degrees C, BTCI shows a continuous exchange curve which indicates that the exchange mechanism is of the EX2 type and no detectable conformational changes occur in the protein. Deviations from this exchange curve are found at pD 7.3 and 8.0. About 60% of the peptide hydrogens of BTCI are exchanged for delta Go less than or equal to 2 kcal/mol, and 90% for delta Go less than 6 kcal/mole. For reduced and carboxymethylated BTCI, exchange data suggest a much more open conformation in comparison with the unmodified protein. However, some residual structure appears to be maintained, after scission of the disulfide bonds. The exchange data indicate that, as a consequence of the formation of the beta-trypsin-BTCI complex, part of the peptide groups of the enzyme and/or inhibitor become less accessible to the isotopic exchange.
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PMID:Hydrogen-deuterium exchange in the black-eyed pea trypsin and chymotrypsin inhibitor and its complex with beta-trypsin. 721 61

Amino acid analysis of oxidized or reduced and carboxymethylated beta-glucuronidase have shown the presence of 24 cysteic acid or S-carboxymethylcysteine residues respectively per mole of the tetrameric enzyme. Titration of sulfhydryl groups gave eight cysteine residues, and by difference 16 half-cystine residues per mole. Six peptides containing radiolabelled cysteine residues were isolated from pepsin and chymotrypsin digest of reduced and S-carboxymethylated beta-glucuronidase by ion-exchange chromatography or gel filtration, followed by paper ionophoresis and paper chromatography. The peptides were analysed for amino acids and sequenced by the dansyl-Edman procedure. Peptides containing cysteic acid were selectively recovered from thermolysin digests of performic acid-oxidized glucuronidase. The amino acid sequences confirmed that there were only six different peptide sequences containing either cysteine or half-cystine residues in the tetrameric enzyme, supporting the presence of four identical subunits. These sequences wer: (A)-Val-Asx-Val-Ile-Cys-Val-Asx-Ser-Tyr- (B)-Gly-Asx-Leu-Cys-Ser-Gly- (C)-Phe-Val-Val-Ile-Asx-Glx-Cys-Pro-Gly-Val-Gly- (D)-Val-Val-Cys-Leu- (E)-Gln-Ser-Gly-Cys-Leu-Val-Lys-Gly-Tyr- (F)-Cys-Asp-Arg-Tyr-Gly-Ile-Val-Val-.
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PMID:Amino acid sequences containing cysteine or half-cystine residues in beta-glucuronidase. 721 58

The structure of the conjugate of Bowman-Birk soybean proteinase inhibitor (BBI) with the block copolymer of ethylene oxide and propylene oxide (proxanol) containing five moles of proxanol per mole of protein, has been studied. Data from reverse phase hydrophobic HPLC suggest that the conjugate is less hydrophobic compared to native BBI. A shift of the second derivative UV absorption spectrum for the conjugate towards the shortwave region indicates a greater accessibility of the Tyr-59 residue localized in the interdomain region of the BBI molecule for the solvent. It has been assumed that the conjugate-induced increase in Ki for chymotrypsin may be due to both disturbances in the intact structure of the interdomain region of BBI and screening of the anti-chymotrypsin reactive center as a result of hydrophobic interactions of propylene oxide blocks of proxanol with exposed hydrophobic groups around the reactive center. Supporting evidence in favour of BBI molecule hydrophilization as a result of modification by proxanol can be derived from decreased conjugate penetration into intestinal epithelial cells as well as from the slow elimination of the conjugate from mouse blood stream.
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PMID:[Structure and biological properties of a conjugate of Bowman-Birk type soy proteinase inhibitor with a block copolymer of ethylene oxide and propylene oxide]. 777 75

Bovine lens aldose reductase (ALR2) is readily modified by glutathione disulphide (GSSG) to an enzyme form (GS-ALR2) exhibiting a reduced catalytic efficiency with all the substrates tested and a reduced susceptibility to inhibition. The modification, which is completely reversed by reduced glutathione (GSH) or dithiothreitol occurs by a pseudo-first-order process with respect to the enzyme and a second order rate constant of 30 +/- 0.1 mol-1 min-1 at 25 degrees C was determined. By measuring the residual activity of ALR2 incubated in different glutathione redox buffers at 25 degrees C, an apparent redox equilibrium constant of 1.4 +/- 0.1 was evaluated. Thus the rate and the maximal extent of ALR2 inactivation are proportional to the redox ratio of the thiol used as modifying agent (i.e. [GSH]/[GSSG]). The stoichiometric reversibility of the enzyme modification might be impaired by a reduced solubility of GS-ALR2 with respect to ALR2 and by an increased susceptibility of the modified enzyme to proteolysis. While the native enzyme form is rather insensitive to proteolytic breakdown. GS-ALR2 is easily degraded by chymotrypsin with the generation of a peptide of 26 kDa with an aminoacid sequence at the aminoterminal side compatible with proteolysis at level of Tyr 7 of aldose reductase. A reduced efficiency in the enzyme-cofactor binding following the GSSG dependent modification of ALR2, appears to be associated to the thiol accessibility of GS-ALR2 measured at different temperatures. GS-ALR2 is characterized by the presence of one glutathione residue, linked through a mixed disulphide bond. This is sustained by: (i) the isoelectric point for the modified enzyme of 4.75, which is 0.1 pH units lower than that observed for the native enzyme, which indicates the contribution of an acidic residue to the pI of GS-ALR2; (ii) the incorporation of radioactivity coming from [3H] labelled GSSG accounting for the presence of one equivalent of glutathione per mole of enzyme. Besides being a general feature of protein reactivity in oxidative conditions, the glutathione-mediated ALR2 modification might be part of a cell strategy to preserve reducing power in conditions of oxidative stress.
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PMID:Glutathione dependent modification of bovine lens aldose reductase. 792 85

Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide. The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride. The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction. At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions. Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis. Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation. Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin. The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.
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PMID:Synthesis, characterization and properties of sialylated catalase. 865 33

Liver fatty acid binding protein (L-FABP) appears to contain several different forms that may result from post-translational modification or bound ligand. To further assess this possibility, L-FABP was purified from rat liver homogenate and two putative isoforms separated using a sulfonyl column, a strong cation exchange resin. Fraction I eluted at 0.2 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 98% L-FABP. Fraction II eluted at 1.0 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 99% L-FABP. Both fractions contained approx. 0.15 moles of endogenous bound fatty acid per mole of protein, while L-FABP not subjected to the cation exchange step contained 0.75 moles of fatty acid per mole of protein. Fractions I and II had a greater proportion of saturated and monounsaturated fatty acids with a large reduction in polyunsaturated fatty acids compared to L-FABP not fractionated by cation exchange. Mass spectral analysis indicated the molecular mass of Fraction I was 14,315.02 +/- 0.35 Da and Fraction II was 14,315.86 +/- 0.34 Da. The peptide map for each fraction was determined by limited digestion of each fraction with either trypsin, Asp-N, or chymotrypsin to yield overlapping peptide fragments. Mass spectral analysis of these digests indicated the two proteins had identical amino acid fragments and that Cys69 was reduced and there were no Asn to Asp exchanges. Hence, these two forms of L-FABP were not isoforms and were not the result of differences in bound fatty acid. It is proposed that these two distinct forms of rat L-FABP were structural conformers based on two alternative folding pathways.
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PMID:Isolation and characterization of two distinct forms of liver fatty acid binding protein from the rat. 998 72

Human kallikrein 2 (hK2) is a serine protease expressed predominantly in the prostate which has 80% homology to prostate-specific antigen (PSA). hK2 is an active trypsin-like protease which has been shown by immuno-histochemical staining to be more highly expressed in prostate carcinoma than in benign prostate tissue. Unlike PSA, hK2 activates pro-PSA , pro-hK2 and the zymogen form of urokinase-type plasminogen activator (uPA), an extracellular protease correlated with prostate cancer and metastasis. We show here that hK2 rapidly forms a complex with plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of uPA in tissues. In addition, hK2 inactivated 6 to 7 mol of PAI-1 by cleavage at Arg346-Met347 for every mole of hK2-PAI-1 complex formed. In contrast with hK2, PSA neither complexed with nor inactivated PAI-1. PAI-1 inhibited hK2 comparably with protein C inhibitor (PCI) and at least 20 times more rapidly than alpha1-anti-chymotrypsin (ACT). N-Terminal sequencing shows that hK2 forms a covalent complex with PAI-1, PCI and ACT after cleavage at Arg346-Met347, Arg354-Ser355 and Leu358-Ser359, respectively. During complex formation, hK2 inactivated PAI-1 but did not inactivate ACT or PCI. Our current results suggest that the increased hK2 expression in prostate cancer tissues could influence cancer biology not only by activation of uPA but also by inactivation of its primary inhibitor, PAI-1.
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PMID:Prostatic human kallikrein 2 inactivates and complexes with plasminogen activator inhibitor-1. 1020 59

We analyzed the energetic importance of residues surrounding the hot spot (the P(1) position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P(1) position to methyl groups caused a decrease in Delta G(den) values up to 6.4 kcal mole(-1), it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole(-1)) than chymotrypsin (decrease up to 3.5 kcal mole(-1)). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P(1)-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain-main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P(1)-S(1) electrostatic interaction.
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PMID:Analysis of serine proteinase-inhibitor interaction by alanine shaving. 1191 24

Preparation of Reversibly Inactivated (R.I.) Phage.- If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5-6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.- The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0 degrees C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.- There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20 degrees C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active right harpoon over left harpoon inactive phage, may be repeated many times at 0 degrees C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except those causing irreversible inactivation, above). The concentration required to prevent R.I. is lower, the higher the valency of either the anion or cation. There are great differences, however, between salts of the same valency, so that the chemical nature as well as the valency is important. Peptone, urea, and the amino acids, tryptophan, leucine, isoleucine, methionine, asparagine, dl-cystine, valine, and phenylalanine, stabilize the system at pH 7, so that no change occurs if a mixture of R.I. and active phage is added to such solutions. The active phage remains active and the R.I. phage remains inactive. The R.I. phage in pH 7 peptone becomes active if the pH is changed to 5.0. This does not occur in solutions of urea or the amino acids which stabilize at pH 7.0. Kinetics of Reversible Inactivation.- The inactivation is too rapid, even at 0 degrees to allow the determination of an accurate time-inactivation curve. The rate is independent of the phage concentration and is complete in a few seconds, even in very dilute suspensions containing <1 x 10(4) particles/ml. This result rules out any type of bimolecular reaction, or any precipitation or agglutination mechanism, since the minimum theoretical time for precipitation (or agglutination) of a suspension of particles in a concentration of only 1 x 10(4) per ml. would be about 300 days even though every collision were effective. Mechanism of Salt Reactivation.- Addition of varying concentrations of MgSO(4) (or many other salts) to a suspension of either active or R.I. phage in 0.01 M, pH 6 acetate buffer results in the establishment of an equilibrium ratio for active/R.I. phage. The higher the concentration of salt, the larger proportion of the phage is active. The results, with MgSO(4), are in quantitative agreement with the following reaction: See PDF for Equation Effect of Temperature.- The rate of inactivation is too rapid to be measured with any accuracy, even at 0 degrees C. The rate of reactivation in pH 5 peptone, at 0 and 10 degrees , was measured and found to have a temperature coefficient Q(10) = 1.5 corresponding to a value of E (Arrhenius' constant) of 6500 cal. mole(-1). This agrees very well with the temperature coefficient for the reactivation of denatured soy bean trypsin inhibitor (Kunitz, 1948). The equilibrium between R.I. and active phage is shifted toward the active side by lowering the temperature. The ratio R.I.P./AP is 4.7 at 15 degrees and 2.8 at 2 degrees . This corresponds to a change in free energy of -600 cal. mole(-1) and a heat of reaction of 11,000. These values are much lower than the comparative one for trypsin (Anson and Mirsky, 1934 a) or soy bean trypsin inhibitor (Kunitz, 1948). Neither the inactivation nor the reactivation reactions are affected by light. The results in general indicate that there is an equilibrium between active and R.I. phage. The R.I. phage is probably an intermediate step in the formation of inactive phage. The equilibrium is shifted to the active side by lowering the temperature, adjusting the pH to 7-8 (except in the presence of high concentrations of peptone), raising the salt concentration, or increasing the valency of the ions present. The reaction may be represented by the following: See PDF for Equation The assumption that the active/R.I. phage equilibrium represents an example of native/denatured protein equilibrium predicts all the results qualitatively. Quantitatively, however, it fails to predict the relative rate of digestion of the two forms by trypsin or chymotrypsin, and also the effect of temperature on the equilibrium.
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PMID:Inactivation and reactivation of B. megatherium phage. 1327 23

1. The reaction of alpha-chymotrypsin with sodium periodate at pH5.0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K(m) values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.
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PMID:THE ROLE OF METHIONINE IN ALPHA-CHYMOTRYPSIN-CATALYSED REACTIONS. 1433 55

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