Pancreatic trypsin inhibitor. 2. Reaction with trypsin

Green, N. M.; Work, Elizabeth

doi:10.1042/bj0540347

Cited by 299 publications

(105 citation statements)

References 9 publications

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“…1: 1 is found on extrapolation to 100% inhibition, indicating that the catalytic domain is responsible for enzyme/inhibitor interaction. The deviation from 100 % inhibition was used to calculate the approximate Kd of enzyme-inhibitor complexes (Green and Work, 1953). The results are summarized in Table 3 CaCI2/150 mM NaCI, pH 7.5, for 30 min at 37°C.…”

Section: Ii-g-p-q-t-p-k-mentioning

confidence: 99%

Fragmentation of human polymorphonuclear-leucocyte collagenase

Knauper

Osthues

DeClerck

et al. 1993

Biochemical Journal

109

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Human polymorphonuclear-leucocyte collagenase (M(r) 64,000) shows autoproteolytic degradation to two major fragments of M(r) 40,000 and M(r) 27,000. N-terminal sequence data and investigation of the substrate specificity of the fragments demonstrate that the M(r)-40,000 fragment corresponds to the catalytic domain, whereas the M(r0-27,000 fragment shows no enzymic activity. The activity profile of the M(r)-40,000 fragment is comparable with the specificity of the intact active collagenase (M(r) 64,000), but the ability to cleave collagen was lost. The enzymic activity of this fragment can be inhibited by either tissue inhibitor of metalloproteinase (TIMP)-1 or recombinant TIMP-2 in a 1:1 molar ratio. The C-terminal part of the enzyme (M(r) 27,000), important for the binding reaction with collagen substrates, is involved in collagenolysis.

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Section: Ii-g-p-q-t-p-k-mentioning

confidence: 99%

Fragmentation of human polymorphonuclear-leucocyte collagenase

Knauper

Osthues

DeClerck

et al. 1993

Biochemical Journal

109

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“…It is consequently an excellent model for the description of the structural properties of mini-proteins. Others studies on miniproteins have been carried out in this laboratory with neurotoxins composed of 63 amino acids [15].The inhibitor reacts with trypsin, one of the best known enzymes [16,17], to form an extremely stable 1 : 1 complex [18,19]. Because of the considerable structural as well as functional knowledge concerning the two partners in the complex and because of the very unusual stability of their association, the interaction between trypsin and its inhibitor appears t o be an excellent model system for the study of effector-receptor association in the protein field.…”

mentioning

confidence: 99%

The Conformational Properties of the Basic Pancreatic Trypsin-Inhibitor

1971

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The conformational properties of the basic pancreatic trypsin-inhibitor have been studied using both physical and chemical techniques.The structural properties of this mini-protein are quite unusual. The folded conformation remains unaltered a t 77 "C and pH 2.1 or in 6 M guanidine-HCI at pH 7.5. The selective cleavage [22] of the disulfide bridge Cys,, -Cys,, reduces considerably the stability of the inhibitor towards heat or guanidine-HC1. A conformational study of the carboxamidomethyl, carboxymethyl, and aminoethyl derivatives of the inhibitor has been carried out after partial reduction and modification of Cys14 and Cys,, by iodoacetamide, iodoacetic acid, and ethyleneimine.There exist three folded isomeric forms of the inhibitor between pH 1 and 11. Form I is the most stable form; it exists near neutral pH. It is extremely thermostable and remains folded in 6 M guanidine-HC1. Form I1 is stable below pH 1.5-2; its transition temperature is 81 "C. Form I11 is predominant at pH 10-11 at 20 "C. A detailed analysis of the conformational isomers I, I1 and I11 has been ca,rried out with the carboxamidomethylated inhibitor.Both physico-chemical and chemical data indicate that the acidic conformational change I + I1 is dependent upon the ionisation of a buried carboxylate which may be the side-chainThe alkaline isomerisation I + I11 is induced by the unmasking of the a-ammonium of the N-terminal arginine residue. The apparent pK of the group is 9.4 a t 25 "C. Acetylation of the a-amino group stabilizes a form very similar to I11 and prevents the formation of form I a t neutral pH.of Glu,.The amino-acid sequence of the basic trypsininhibitor from bovine pancreas is known [l --31 and the structure-function properties of the protein have been studied in several laboratories [4-141. Lysine-15 was shown to be an essential element of the active site [lo].Since this trypsin inhibitor is comprised of only 58 amino acids in a single polypeptide chain it is one of the smallest known proteins. It is consequently an excellent model for the description of the structural properties of mini-proteins. Others studies on miniproteins have been carried out in this laboratory with neurotoxins composed of 63 amino acids [15].The inhibitor reacts with trypsin, one of the best known enzymes [16,17], to form an extremely stable 1 : 1 complex [18,19]. Because of the considerable structural as well as functional knowledge concerning the two partners in the complex and because of the very unusual stability of their association, the interaction between trypsin and its inhibitor appears t o be an excellent model system for the study of effector-receptor association in the protein field.

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“…Canine PST1 inhibited bovine trypsin activity stoichiometrically ( fig.%) and the approximate dissociation constant at pH 8 was below 10m9 M, when the method of Green and Work [14] was used.…”

Section: Trypsin Inhibitory Activity Of Canine Pstimentioning

confidence: 98%

Purification and complete amino acid sequence of canine pancreatic secretory trypsin inhibitor

Kikuchi¹,

Nagata²,

Yoshida³

et al. 1985

FEBS Letters

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Pancreatic secretory trypsin inhibitor (PSTI) was purified from canine pancreatic juice by HPLC. Canine PST1 inhibited bovine trypsin activity stoichiometrically and strongly with a dissociation constant of below 10mg M. The amino acid sequence of canine PST1 was determined by conventional methods. It had one more amino acid residue at the amino-terminus than other mammalian PSTIs, i.e. human, porcine, bovine and ovine.

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Pancreatic trypsin inhibitor. 2. Reaction with trypsin

Abstract: The reactions of trypsin with its inhibitors are among the few known cases of specific interaction between two purified proteins (Kunitz & Northrop, 1936; Kunitz, 1947; Fraenkel-Conrat, Bean &

Cited by 299 publications

References 9 publications

Fragmentation of human polymorphonuclear-leucocyte collagenase

Fragmentation of human polymorphonuclear-leucocyte collagenase

The Conformational Properties of the Basic Pancreatic Trypsin-Inhibitor

Purification and complete amino acid sequence of canine pancreatic secretory trypsin inhibitor

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