Spin-label studies on protein proteinase inhibitors: Complex formation and conformational changes of the bovine trypsin inhibitor (Kunitz)

1981 ◽  
Vol 7 (4) ◽  
pp. 285-285 ◽  
Author(s):  
H. R. Wenzel ◽  
H. Tschesche ◽  
E. Goldammer ◽  
U. Netzelmann
Keyword(s):  
Complex Formation ◽  
Conformational Changes ◽  
Trypsin Inhibitor ◽  
Spin Label ◽  
Proteinase Inhibitors ◽  
Bovine Trypsin

The interaction of ligands with chemically modified phosphorylase b

1976 ◽  
Vol 54 (5) ◽  
pp. 494-499
Author(s):  
D. Brooks ◽  
S. J. W. Busby ◽  
J. R. Griffiths ◽  
G. K. Radda ◽  
O. Avramovic-Zikic
Keyword(s):  
Conformational Changes ◽  
Spin Label ◽  
Native Enzyme ◽  
Carboxyl Groups ◽  
Chemically Modified ◽  
Allosteric Effector ◽  
Spin Resonance ◽  
Label Probe ◽  
Glycine Ethyl Ester ◽  
Modified Enzyme

Phosphorylase b which had been inactivated with 5-diazo-1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein.Phosphorylase b which had been inactivated by carbodiimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose- 1-phosphate.The actions of 5-diazo-1H-tetrazole and carbodiimide on phosphorylase are discussed in the light of the above observations.

Conformational Changes in ι- and κ-Carrageenans Induced by Complex Formation with Bovine β-Casein

2007 ◽  
Vol 8 (2) ◽  
pp. 368-375 ◽  
Author(s):  
Tatiana V. Burova ◽  
Natalia V. Grinberg ◽  
Valerij Ya. Grinberg ◽  
Anatoly I. Usov ◽  
Vladimir B. Tolstoguzov ◽  
...  
Keyword(s):  
Complex Formation ◽  
Conformational Changes

scholarly journals Antiviral proteinase inhibitors of plant and animal origin

2020 ◽  
Vol 2 (2) ◽  
pp. 43-48
Author(s):  
Valentina Divocha ◽  
◽  
Irina Komarevzeva ◽  
Keyword(s):  
Influenza Virus ◽  
Trypsin Inhibitor ◽  
Proteinase Inhibitors ◽  
Animal Origin ◽  
Ether Anesthesia ◽  
The Past ◽  
Activity Of Enzymes ◽  
Academy Of Sciences ◽  
Observation Period

Introduction: Over the past 10 years, much attention has been paid to the development of new antiviral drugs based on the suppression of the proteolytic activity of enzymes by trypsin inhibitors of plant and animal origin. Material and methods: We used a trypsin inhibitor from barley, trielin- (isolated by employees of the Agro-Industrial Institute of Selection and Genetics of the Ukrainian Academy of Sciences from the salivary glands of a dog); ovomukoid (isolated from duck eggs by employees of N, I, Bach Research Institute of Biology, Russian Academy of Sciences); Influenza virus APR 8/34 (fourth passage), adapted to the lungs of mice at a dose of 20 LD /0.1 ml, titre HA( hemagglutenin) 1:32) ,white BALB/c mice weighing 12-14 g. Infection with influenza virus and treatment with inhibitors was carried out intranasally under light ether anesthesia. Doses studied were: 0.5mg/ml; 2.5 mg/ml; 5.0 mg/ml; The treatment regimen of 10 mg/ml differed only in the initial stages (1 hour before infection, during infection and 1 hour after infection, and then 6 hours after infection, 24 hours after infection, 48 hours after infection, 72 hours after infection and 96 hours after infection). Results and discussion: We found that an in vivo inhibitor from barley at a dose of 10 g/l delayed the development of influenza for 8 days. The ovomukoid possessed only prophylactic properties at a dose of 100 gamma / ml. With an increase in dose, it was toxic to animals. Trielin at a dose of 10 g/l had a pronounced therapeutic effect in influenza and was not toxic. The presence of hemagglutinin influenza virus in the lungs of treated mice was observed only on the 10th day after infection; 40% of the animals remained alive for 14 days (observation period).

A Spin-label Study on Calcium-induced Conformational Changes of Troponin Components*

1974 ◽  
Vol 75 (1) ◽  
pp. 211-213 ◽  
Author(s):  
Setsuro EBASHI ◽  
Shun-ichi OHNISHI ◽  
Shin-ichi ABE ◽  
Koscak MARUYAMA
Keyword(s):  
Conformational Changes ◽  
Spin Label ◽  
Label Study

CHARACTERIZATION OFα2-ANTIPLASMIN.REACTIVE SITE VARIANTS PRODUCED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
W E Holmes ◽  
H R Lijnen ◽  
D Collen
Keyword(s):  
Sequence Analysis ◽  
Complex Formation ◽  
Active Site ◽  
Activity Analysis ◽  
Site Directed Mutagenesis ◽  
Thrombin Inhibitor ◽  
Directed Mutagenesis ◽  
Reactive Site ◽  
Antithrombin Activity ◽  
Bovine Trypsin

α2-Antiplasmin (α2AP) is the primary physiological plasmin inhibitor in human plasma. The inhibition is rapid (second order rate constants (k1) are expressed as M−1 s−1 ) (k1 = 2 × 107) and occurs as the consequence of an irreversible 1:1 stoichiometric complex formation; the exact nature of and the forces involved in complex formation are not fully understood. In fact, what makes α2AP an inhibitor, rather than simply a substrate remains unresolved. Recently, we deduced the primary structure of α2 AP from the sequence of its cDNA. 95%of this sequence was confirmed by amino acid (aa) sequence analysis of naturalα2 AP (α2 AP)? The 452 aa molecule contains 2 disulfide bonds and 4 glycosylated Asn residues, aa sequence alignment confirmed α2AP's membership in the Serpin family. The reactive site sequence as determined by NH2 - and COOH-terminal aa sequence analysis of the plasmin-modified inhibitor and the released M−r ∼ 8000 peptide is Met362-Ser363-Arg364-Met365-Ser366, P3-P2-P1-P'1-P'2, respectively.Natural and engineered P1 residue substitutions in the Serpin α2 -antitrypsin ( α2 AT) have shown altered specificities and efficiencies. To further examine the role of P and P' residues in determining Serpin specificity, in the present study we have by site-directed mutagenesis, deleted (△) the P'l-Met365 residue of a AP thereby producing a recombinant (r) inhibitor (r α2 AP△Met365) whose putative new reactive site mimics that of antithrombin III (ATIII) and a AT-Pittsburgh (Pl-Arg-P'1-Ser). A second variant was constructed (ra2AP△Arg364) in which the Pl-Arg364 residue was deleted, producing the new sequence Met362-Ser363-Met364-Ser365, containing 2 potential sites analogous to the Pl-P'l, Met-Ser reactive site of α2 AT. The variants and r α2 AP were expressed in CH0 cells, purified and compared with n α2 AP, α2AT and ATIII for the ability to inhibit plasmin, thrombin, trypsin and elastase. n α2 AP and r α2 AP had nearly identical inhibition constants and like ATIII did not inhibit neutrophil elastase. Without heparin both α2 APs and ATIII inhibited thrombin moderately (k1 = 2 to 4× 103 ). Bovine trypsin was neutralized by the α2 APs with k1 = 3 × 106 and by ATIII with k1 = 1 × 105. The α2APs inhibited plasmin (k1 = 2 ×107 ) much more efficiently than ATIII (K1 =2 × 103 ). In contrast, was a highly effective antielastase (k1 = 1 × 107 ), a poor plasmin and thrombin inhibitor ancl inhibited bovine trypsin with = 2 × 10. As reported by others, α2 AT-Pittsburg has greatly reduced antielastase activity and greatly enhanced antithrombin activity. Analysis of ra APAMet365 revealed little change in activity toward plasmin, trypsin and elastase. Thus, α2 AP has no absolute requirement for Met .in the P'l position in order to effectively inhibit plasmin and trypsin. The other P^ subsites appear to be spatially flexible as deletion of the natural P'l residue must displace them. Contrary to prediction a 20-fold decrease in antithrombin activity was observed rather than an enhanced activity. Analysis of rα2 AP△Arg364 showed that it is unreactive with plasmin, trypsin and thrombin, but that it has acquired a significant antielastase activity (k1 = 1.5 × 105). The exact PI residue(s) has not been determined but removal of the bulky basic Arg364 may have resulted in accessibility of the predicted reactive site(s) peptide bond(s) Met362-Ser363 or Met364-Ser365 to the active site cleft of elastase. α2AP'Enschede', a natural mutant with deficient antiplasmin activity, was shown to contain an Ala insertion between aa 353 and 357, 7 to 10 positions NH2-terminal to its reactive site (Holmes et al., this meeting). This mutation results in conversion of α2 AP'Enschede' from an inhibitor to a substrate that retains a high affinity for the active site of plasmin.

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