Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Any research which has shed light on the nature of disease or opened new ways to its prevention or cure is here termed relevant, and the question will be asked whether the research could have been planned with these aims in mind. Examples will be taken from the chemistry and X-ray analysis of proteins and from molecular genetics. Blow and Hartley determined the amino acid sequence and atomic structure of the digestive enzyme chymotrypsin in order to solve the problem of enzymic catalysis. They succeeded but what they found has proved to be of much wider improtance than could have been foreseen at the outset: it gives the key to the mechanism of blood clotting and suggests new methods for its control. X-ray analysis of haemoglobin was started at a time when the structure of proteins was regarded as the central problem of biology, but it did not seem likely then that the results would shed light on the molecular pathology of inherited diseases. Ames made a life-long study of the genetic control of histidine biosynthesis in Salmonella because it represents an example of a widely used biological mechanism, but without expecting it to have any practical applications. Yet his recent exploitation of the system for the rapid and sensitive detection of chemical carcinogens may represent a breakthrough in cancer prevention. This unpredictable relationship of molecular biology to medicine is symptomatic of the subject's youth.
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PMID:Fundamental research in molecular biology: its relevance to medicine. 80 69

An improved 2.5-A electron density map of chymotrypsinogen was calculated by incorporating heavy-atom anomalous scattering effects and a new model of the molecule was constructed. Phases from x-ray structure factors (R = 0.43) computed from this model were then used in the calculation of another electron density map against which the model was further refined. The catalytic Ser-195 side chain in the new model is in the "down" or "acyl" orientation and its Ogamma atom is in position to form a normal hydrogen bond with Nepsilon2 of His-57. In contrast, the corresponding hydrogen bond in alpha-chymotrypsin (Birktoft, J.J., and Blow, D.M. (1972), J.Mol. Biol. 68, 187) is severely distorted, probably as a consequence of a 1.5-A shift in the relative positions of the two cylindrical folding domains composing most of the molecule. We suggest that this activiation induced distortion of the charge-relay, hydrogen-bonding system plays an important role in the genesis of enzymic activity, in accord with an earlier proposal by Wang concerning the role of bent hydrogen bonds in enzyme catalysis (Wang, J.J. (1970), Proc. Natl. Acad. Sci. U.S.A. 66, 874).
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PMID:A detailed structural comparison between the charge relay system in chymotrypsinogen and in alpha-chymotrypsin. 97 71

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.
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PMID:Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae. 132 40

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.
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PMID:Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme. 230 34

Nine substances (Pancrease, Viokase, pork pancreatin, bromelain, papain, cranberry juice, Coca-Cola, chymotrypsin, and distilled water) were tested every half-hour for 4 hr using 900 mm of water pressure to determine their effectiveness in declogging small-bore feeding tubes. At the end of 4 hr the tubes were irrigated with air using a 50-cc syringe. None of the substances tested were effective within 4 hr. Three substances allowed for successful syringe irrigation at the end of 4 hr. They were: chymotrypsin, papain, and distilled water.
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PMID:Declogging small-bore feeding tubes. 312 31

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enzymatic properties of proteolytic derivatives of human alpha-thrombin. 337 50

Thermal inactivation of Berne virus proceeded at a linear rate between 31 degrees and 43 degrees C. Storage at temperatures lower than -20 degrees C preserved the infectivity, while at 4 degrees C appreciable loss occurred between 92 and 185 days. Freeze-drying or desiccation at 22 degrees C caused only insignificant loss of infectivity. Virus preparations were not affected by pH values between 2.5 and 10.3. Inactivation by UV occurred more rapidly than with herpes, toga and rhabdoviruses. Berne virus infectivity was sensitive to pronase and B. subtilis proteinase. It was not inactivated by trypsin and chymotrypsin treatment, which resulted in enhancement of infectivity; low concentrations of pronase (less than 10 micrograms ml-1) had a similar effect on Berne virus. Neither phospholipase C or RNase, alone or in combination, nor sodium deoxycholate (0.1%) inactivated the virus; in contrast, Triton X-100 (0.1%; 1.0%) caused rapid inactivation with a constant level of residual infectivity.
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PMID:Resistance of Berne virus to physical and chemical treatment. 351 25

A component of the cell walls of certain enteric bacteria has been identified that cross-reacts with an HLA-B27-associated cell-surface structure on lymphocytes and other cell types from patients with ankylosing spondylitis. This component, or "modifying factor," from one particular organism, Klebsiella K43-BTS1, has been studied in detail. A purification scheme has been developed using preparative electrofocusing and gel-permeation high performance liquid chromatography techniques and the purified material used in various characterization studies. A previous study demonstrated that the modifying factor has an approximate molecular weight of 30,000 and an isoelectric point of 5.4-5.5. In this study two-dimensional gel electrophoresis experiments demonstrated that the modifying factor is associated with a single protein component of the cell wall of this organism. Pronase and papain destroyed the modifying factor activity whereas trypsin and alpha-chymotrypsin degraded the factor into smaller fragments without destroying its ability to modify B27+ AS- lymphocytes. Neuraminidase did not affect the modifying factor itself but did affect B27+ AS- lymphocytes such that they became unresponsive to modification. Sugar inhibition studies suggested that sugar groups are probably not involved in the function of the modifying factor. The availability of purified modifying factor should permit more detailed chemical analyses as well as functional studies to determine the significance of this molecule to the pathogenesis of ankylosing spondylitis.
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PMID:Biochemical studies on a factor isolated from Klebsiella K43-BTS1 that cross-reacts with cells from HLA-B27 positive patients with ankylosing spondylitis. 353 95

The contributions of various regions of human alpha-thrombin to the formation of the tight complex with hirudin have been assessed by using derivatives of thrombin. alpha-Thrombin in which the active-site serine was modified with diisopropyl fluorophosphate was able to bind hirudin, but its affinity for hirudin was decreased by 10(3)-fold compared to unmodified alpha-thrombin. Modification of the active-site histidine with D-Phe-Pro-Arg-CH2Cl resulted in a form of thrombin with a 10(6)-fold reduced affinity for hirudin. gamma-Thrombin is produced by proteolytic cleavage of alpha-thrombin in two surface loops corresponding to residues 65-83 and 146-150 in alpha-chymotrypsin [Berliner, L. J. (1984) Mol. Cell. Biochem. 61, 159-172; Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. The gamma-thrombin-hirudin complex had a dissociation constant that was 10(6)-fold higher than that of alpha-thrombin. Treatment of alpha-thrombin with pancreatic elastase resulted in a form of thrombin only cleaved in the loop corresponding to residues 146-150 in alpha-chymotrypsin, and this form of thrombin had only a slightly reduced affinity for hirudin. By using limited proteolysis with trypsin, it was possible to isolate beta-thrombin which contained a single cleavage in the loop corresponding to residues 65-83 in alpha-chymotrypsin. This form of thrombin had a 100-fold decrease in affinity for hirudin. Kinetic analysis of the binding of hirudin to beta-thrombin indicated that the 100-fold decrease in affinity was predominantly due to a decrease in the rate of association of the two molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of regions of alpha-thrombin involved in its interaction with hirudin. 366 13

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.
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PMID:Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin. 381 86


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