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W Ardelt

Publications and source records attributed to W Ardelt.

At least 37 records · Page 2Linked to original sources
PubMed1987

Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution.

The molecular structure of the complex between bovine pancreatic alpha-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 A (1 A = 0.1 nm) resolution shell [I greater than or equal to sigma (I)]. The reactive site loop of OMTKY3, from Lys13I to Arg21I (I indicates inhibitor), is highly complementary to the surface of alpha-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 A with 21 residues of the enzyme. A short contact (2.95 A) from O gamma of Ser195 to the carbonyl-carbon atom of the scissile bond between Leu18I and Glu19I is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P'3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the alpha-chymotrypsin portion of this complex with the several structures of alpha and gamma-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common alpha-carbon atoms averages 0.38 A). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central alpha-helix and flanking small 3-stranded beta-sheet) that have alpha-carbon atoms fitting to within 1.0 A (root-mean-square deviation of 0.45 A) whereas the residues of the reactive-site loop differ in position by up to 1.9 A (C alpha of Leu18I). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and alpha-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.

Animals↗
PubMed1987

Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues.

Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron.

Amino Acid Sequence↗
PubMed1985

Turkey ovomucoid third domain inhibits eight different serine proteinases of varied specificity on the same ...Leu18-Glu19 ... reactive site.

We show that eight different serine proteinases--bovine chymotrypsins A and B, porcine pancreatic elastase I, proteinase K, Streptomyces griseus proteinases A and B, and subtilisins BPN' and Carlsberg--interact with turkey ovomucoid third domain at the same Leu18-Glu19 peptide bond, the reactive site of the inhibitor. Turkey ovomucoid third domain was converted to modified (the reactive site peptide bond hydrolyzed) form as documented by sequencing. Complexes of all eight enzymes both with virgin and with modified inhibitor were prepared. All 16 complexes were subjected to kinetically controlled dissociation, and all 16 produced predominantly virgin (greater than 90%) inhibitor, thus proving our point. During this investigation, we found that both alpha-chymotrypsin and especially S. griseus proteinase B convert virgin to modified turkey ovomucoid third domain, even in the pH range 1-2, a much lower pH than we expected. We have also measured rate constants kon and kon* for the association of virgin and modified turkey ovomucoid third domain with several serine proteinases. The kon/kon* ratio is 4.8 X 10(6) for chymotrypsin, but it is only 1.5 for subtilisin Carlsberg. A number of generalizations concerning reactive sites of protein proteinase inhibitor are proposed and discussed.

Amino Acid Sequence↗
PubMed1985

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3).

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].

Animals↗
PubMed1983

Thermodynamics and kinetics of the hydrolysis and resynthesis of the reactive site peptide bond in turkey ovomucoid third domain by aspergillopeptidase B.

1. Aspergillopeptidase B rapidly hydrolyses the -Leu18-Glu19-reactive site peptide bond in turkey ovomucoid third domain (OMTKY3) within the pH-range of 4.0-8.4. The reaction proceeds to equilibrium between OMTKY3 and its modified form with the reactive site peptide bond cleaved (OMTKY3). 2. The dependence of the equilibrium constant (Khyd) on pH indicates that hydrolysis of the reactive site peptide bond apparently does not perturb the pK-values of any preexistent ionizable groups in OMTKY3. 3. The obtained Khyd0 value indicates that free energies of OMTKY3 and OMTKY3 are essentially the same. 4. Hydrolysis of the reactive site peptide bond by aspergillopeptidase B at neutral pH is about 60 times faster than the same reaction catalyzed by subtilisin (Carlsberg), the enzyme strongly inhibited by OMTKY3. 5. Resynthesis of the reactive site peptide bond at neutral pH catalyzed by aspergillopeptidase B (reverse reaction) is almost four orders of magnitude faster than the forward reaction.

Animals↗
PubMed1982

[The radiofibrinogen test and the fibrinsplit product test in the diagnosis of deep veinous thrombophlebitis during radiotherapy. A contribution to the evaluation for silent acute thrombophlebitis (author's transl)].

The radiofibrinogen test is especially useful for the diagnosis of deep veinous thrombophlebitis in the legs in patients undergoing radiotherapy for gyanecological cancer. In 62 patients, 6 deep veinous thrombophlebitis were observed during 124 radium insertions in conjunction with preventive administration of anti-coagulants. The most important test for the early coagulation physiologic diagnosis of deep leg thrombophlebitis is the test for fibrinogen and fibrinsplit products. A suitable procedure is the Latex-agglutination test which is technically uncomplicated. If deep leg vein thrombophlebitis is found by the radiofibrinogen test, the fibrinsplit products always show at least a fourfold increase.

Aged↗
PubMed1982

Refined crystal structure of the molecular complex of Streptomyces griseus protease B, a serine protease, with the third domain of the ovomucoid inhibitor from turkey.

We have determined the crystal structure of the molecular complex between Streptomyces griseus protease B (SGPB), a bacterial serine protease, and the third domain of the ovomucoid inhibitor from turkey. Restrained-parameter least-squares refinement of the structure with the 1.8-A intensity data set has resulted in an R factor of 0.125. The carbonyl carbon atom of the reactive bond between Leu-18 and Glu-19 in the inhibitor lies at a distance of 2.71 A from the O gamma atom of the nucleophilic Ser-195 in SGPB; this distance is 0.5 A shorter than a normal van der Waals contact. Unlike the reactive bond in the pancreatic trypsin inhibitor complexed with bovine trypsin, the Leu--Glu bond of the ovomucoid inhibitor is not distorted from planarity towards a pyramidal configuration.

Animals↗
PubMed1979

[Side effects of drug for thromboembolism prophylaxis. Results from the University Women's Clinic Freiburg/Br].

It is reported on the side-effects of the antithrombotic prophylaxis in 10611 gynecological operations and caesarean sections at the University Hospital Freiburg/Br. Because of multiple side-effects a risk-free method of antithrombotic prophylaxis does not exist. The main problem in using heparin and oral anticoagulants are the complications caused by bleeding. Two cumarine necroses were observed. Two severe anaphylactoid reactions were seen under dextran prophylaxis resulting in the death of one newborn. Wound healing is delayed under low-dose heparin due to an increase in hematomas. Additionally two fatal pulmonary embolisms were caused by the so-called "rebound effect" after use of low-dose heparin.

Adult↗
PubMed1979

Specificity of elastases: degradation of the oxidized beta-chain of insulin by porcine pancreatic elastase II and dog leucocyte elastase.

Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).

Amino Acid Sequence↗
PubMed1977

[Cyclic adenosine monophosphate, prostaglandin E and aggregation of blood platelets in man].

The relationship of cyclic AMP to platelet function has been examined directly by assay of the platelet content of the nucleotide and platelet aggregation. Platelet aggregation is favored by an decrease in platelet cyclic AMP and inhibited by an increase in cyclic AMP. The platelet aggregation is accompanied by reduction in cyclic AMP. beta adrenergic substances increase platelet cyclic AMP in vivo and simultaneously block platelet aggregation. Thrombin selectively causes human platelets to form and release PGE. The amount formed was proportional to the amount of thrombin added and high enough to influence platelet aggregation in vitro. Platelet prostaglandin formation by washed platelets and platelet-rich plasma was not proportional to the degree of platelet aggregation induced by stirring. We were unable to detect PGE-formation in response to ADP. Thrombin-induced synthesis of PGE is inhibited by ADP. Aspirin selectively inhibits PGE-production in human platelets. This reaction does not depend on the extend of platelet aggregation. These studies suggest that cyclic AMP is the key to regulation of platelet aggregation and that PGE, formed by platelets, through its effects on cyclic AMP may play a role in regulating platelet aggregation.

Aspirin↗
PubMed1976

Neutral elastolytic proteinase from canine leucocytes. Purification and characterization.

1. A neutral proteinase (EC 3.4.-.-) with elastolytic activity was isolated from canine bloodstream leucocytes, and purified to apparent homogeneity by a two-step procedure consisting of DEAE-Sephadex chromatography and molecular sieving on Sephadex G-75. 2. The molecular weight of the enzyme was 23 500, and the absorbance (A1%1cm) at 282 nm was 6.1. Amino acid analysis showed high content of glycine, aspartic acid, and valine, and low proportion of methionine, lysine and histidine as well as the absence of tyrosine in the enzyme molecule. 3. The proteinase was active against several protein substrates as well as towards N-t-butyloxycarbonyl-L-alanine p-nitrophenyl ester, N-acetyl-L-alanyl-tyrosine ethyl ester. 4. The enzyme was inactivated by diisopropylfluorophosphate, N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone, and N-p-tosyl-L-phenylalanine chloromethyl ketone. Inhibition by some natural proteinase inhibitors was also noted.

Amino Acids↗