Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When N-[14C] acetylbenzotriazole, presented here as a new agent for the acetylation of proteins, reacted at pH 8 and 25 degrees C with delta-chymotrypsin, 15 amino groups (the epsilon-amino groups of lysing residues and the alpha-amino terminus of half-cystine-1) and two phenolic groups (those of the two exposed tyrosine residues) were acetylated with respective pseudo first-order constants of 0.056 +/- 0.003 and 0.15 +/- 0.03 min(-1). Surprisingly, in contrast with the acetic anhydride reaction, the alpha-amino group of Ile-16 was found to be not acetylated as shown by N-terminus determination and activity measurements: the modified delta-chymotrypsin (or acetylated delta-chymotrypsin) was fully active after neutral dialysis. Only a transient inactivation due to the incorporation of one [14C] acetyl group per mole of catalytic site was observed. The kinetic constant found for reactivation at pH 8.5 was 0.315 +/- 0.005 min(-1) at 25 degrees C. The enzyme-catalyzed hydrolysis of N-acetylbenzotriazole was described by a k(cat) value of 0.093 +/- 0.005 min(-1) at pH 7 and 25 degrees C. Circular dichroism changes observed at 230 nm during the reaction at pH 8.5, of acetylated delta-chymotrypsin with N-acetylbenzotriazole indicated a total conversion of the amount of enzyme molecules which were in the 'inactive' or 'alkaline' conformation at this pH, into the 'active' or 'neutral' one. Benzotriazole alone was unable to induce such a conformational change. The rate constant of the reverse structural process from the 'neutral' to the 'alkaline' conformation was 0.32 +/- 0.02 min(-1): identical to that of the deacetylation of the catalytic site. Thus, the unusual lack of acetylation of Ile-16 alpha-amino group during delta-chymotrypsin treatment with N-acetylbenzotriazole is interpreted as a stabilization of the enzyme 'neutral' conformation where the Ile-16 alpha-amino group is buried, thus inaccessible to the reagent. The properties of the delta-chymotrypsin modification using N-acetylbenzotriazole led to practical uses: direct spectrophotometric titration of chymotrypsin operational normality at pH 7 and rapid preparation of acetylated delta-chymotrypsin. As a protein reagent, N-acetylbenzotriazole is particularly interesting because of its reactivity towards amino and phenolic groups of amino acid residues, its stability at acid pH, i.e., k(hydrolysis=7.38 X 10(-3) min(-1) at 25 degrees C [Ravaux et al. (1971) Tetrahedron Letters, 4013-4015] and its aromaticity, responsible for optical properties.
 ...
PMID:N-Acetylbenzotriazole as a protein reagent. Specific behaviour towards delta-chymotrypsin. 0 80

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.
 ...
PMID:Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors. 4 22

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.
 ...
PMID:Purification and characterization of a trypsin inhibitor from Solanum tuberosum. 24 76

An enzyme that catalyzes the conversion of 2-amino-6-(5'-triphosphoribosyl)amino-5- or 6-formamido-6-hydroxypyrimidine, but not of guanosine triphosphate, to quinonoid 6-(D-erythro-1'-2'-3'-trihydroxypropyl)dihydropterin triphosphate and formic acid has been purified to homogeneity from some mammalian brain and liver. The enzyme of a single strand is a basic protein of 9177 daltons consisting of 68 amino acid residues--except the enzyme from rat brain, which has one additional aspartic acid as residue 7. The enzyme possesses three free SH groups and, in its most active form, 1 mol of phosphate per mole of enzyme. Peptides isolated after hydrolysis with trypsin, chymotrypsin, or weak acid were separated by thin-layer chromatography and sequenced manually by Edman degradation. The complete sequence of the molecule was established as follows: (formula: see text)
 ...
PMID:Biopterin. VI. Purification and primary amino acid sequence of mammalian D-erythro-7,8-dihydroneopterin triphosphate synthetase. 49 48

Alpha-crystallin exhibits variable inhibition of several members of the chymotrypsin family of proteinases. Complete inhibition of elastase was obtained by the addition of either alpha-crystallin or a sonicated preparation of the water-insoluble fraction from bovine lens. Little or no inhibition was seen, however, with either beta-crystallin or bovine serum albumin under the same conditions. Complete binding of elastase was demonstrated by Sephadex G-100 gel filtration chromatography, and a direct correlation between binding and inhibition was obtained. This observation permitted us to do a Scatchard analysis of the inhibition data. Scatchard plots for the binding of elastase gave a biphasic response suggesting two separate binding sites. These sites had Kd values of 15 and 40 nM for alpha-crystallin and 6 and 42 nM for the bovine water-insoluble fraction. Similarly, a Dixon plot exhibited a Ki value of 3 nM and was consistent with non-competitive inhibition. One mole of alpha-crystallin (8 x 10(5) Da), or an equivalent amount of water-insoluble protein, bound from 13 to 19 mol of elastase which were about equally divided between the higher and lower affinity sites. Saturation studies confirmed 20 and 16 elastase binding sites per 8 x 10(5) Da for alpha-crystallin and water-insoluble protein, respectively. DFP-elastase was capable of binding to alpha-crystallin suggesting that a proteolytic cleavage was not required for complex formation. Stability measurements showed a linear return to 60% of the original activity over a 30-min period. Therefore, the interaction between elastase and alpha-crystallin resembles that of a heterologous protease:inhibitor complex in both binding and stability.
 ...
PMID:Characterization of the elastase inhibitor properties of alpha-crystallin and the water-insoluble fraction from bovine lens. 154 28

The reaction between the three Bowman-Birk proteinase inhibitors isolated from fenugreek seeds (TFI-B2, TFI-N2 and TFI-A8) and the human and bovine proteinases was investigated by studying the complexes formed and their properties. TFI-B2, the Lys-Leu trypsin chymotrypsin inhibitor, can bind 1.9 mol human trypsin (HT), 1.3 mol bovine trypsin (BT) and/or 0.4 mol human (HCT) or bovine (BCT) chymotrypsin per mole of inhibitor. HT was bound at the two reactive sites and BT mainly at the lysine-containing trypsin-reactive site, whereas HCT and BCT were only bound at the leucine-containing chymotrypsin-reactive site. TFI-N2, the Arg-Leu trypsin chymotrypsin inhibitor, could bind 1 mol BT and BCT, but 1.3 mol HT and 1.2 mol HCT per mole of inhibitor. In addition to the usual binding, the human enzymes could also be bound at the respective "wrong" reactive site. TFI-A8, the Arg-Arg trypsin inhibitor, binds 2 mol HT or BT per mole of inhibitor at the two trypsin-reactive sites, whereas HCT and BCT (about 0.2 mol/mol) are bound to one of the two "wrong" reactive sites.
 ...
PMID:Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reaction with the human and bovine proteinases. 176 94

A protease inhibitor specific to trypsin and chymotrypsin was purified from horsegram (Dolichos biflorus) with the inhibition index 0.24 micrograms/micrograms for trypsin and 0.36 micrograms/micrograms for chymotrypsin. In SDS-PAGE, the inhibitor protein was seen as a single band with apparent molecular mass Mr = 15,500. However, on fast protein liquid chromatography (FPLC) or non-denaturating PAGE, the inhibitor resolved into four components revealing the existence of isoinhibitors. Data on amino acid analysis indicate that the isoinhibitors are closely related. The major amino acids in the inhibitor are half cystine (18.9 mole %), aspartic acid (12.7 mole %) and serine (14.3 mole %). The inhibitor was partially stable to 0.1% sodium dodecyl sulphate, 8M urea or 6M guanidine hydrochloride. The inhibitory activity was lost on reduction or carboxamidomethylation or acetylation. Modification of the arginine groups or CNBr cleavage of the protein did not result in significant loss of either tryptic or chymotryptic inhibitory activities. The isoinhibitors separated by FPLC reacted with polyclonal antibody raised in rabbits and had pI values ranging from 4.8-5.1. The horsegram inhibitor thus resembles other Bowman-Birk protease inhibitors.
 ...
PMID:Nature of the tryptic/chymotryptic inhibitor from horsegram (Dolichos biflorus). 181 76

A calsequentrin (CS)-like glycoprotein is present in the sarcoplasmic reticulum (SR) of chicken pectoralis muscle, which displays unusual properties: it binds relatively low amounts of Ca2+, compared to CS in mammalian skeletal muscle (Yap & MacLennan, 1976), it does not exhibit a marked pH-dependent shift in mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and its metachromatic staining properties with Stains All are likewise peculiar (Damiani et al., 1986). We have now definitively localized the same protein to the junctional terminal cisternae (TC) fraction of the SR of chicken pectoralis muscle and have further characterized it, following purification by crystallization with Ca2+ and by Ca2(+)-dependent elution from phenyl-Sepharose columns. The purified protein (apparent Mr: 51 kDa), isoelectrofocuses at pH 4.5, and is readily identified on blots by a 45Ca overlay technique, similar to CS of rabbit skeletal muscle, but it binds half as much Ca2+ (about 20 moles of Ca2+ per mole of protein), as estimated by equilibrium dialysis. However, the chicken protein shares extensive similarities with mammalian CSs, concerning Ca2(+)-induced changes in maximum intrinsic fluorescence and the Ca2(+)-modulated interaction with phenyl-Sepharose, as well as in being protected by Ca2+ from proteolysis by either trypsin or chymotrypsin. We discuss how the presence of a Ca2(+)-regulated hydrophobic site in the CS molecule appears to be the most invariant property of the CS-family of Ca2(+)-binding proteins.
 ...
PMID:Characterization of calsequestrin of avian skeletal muscle. 235 47

Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin. 301 7

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.
 ...
PMID:Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds. 308 4


1 2 3 4 Next >>