alpha-chymotrypsin and 3-4-dichloroisocoumarin

Polyacrylamide gel electrophoresis and high performance liquid chromatography of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary, lung, and liver showed marked differences in the pattern of subunits. The concentrations of LMP7 in the lung and liver were 10 and 5 times, respectively, greater than those in the pituitary, whereas the chymotrypsin-like activity and the amount of a subunit (BO2), necessary for its expression, were markedly decreased in the lung and moderately decreased in the liver. Lower trypsin-like, small neutral amino acid preferring, and peptidyl-glutamyl-peptide hydrolyzing activities were also found in the lung and liver. The activity of the branched chain amino acid preferring component (BrAAP), predominantly latent in the pituitary, was highly activated in the lung and liver, as evidenced by a greatly decreased Km and a 20-fold increase of the specificity constant Vmax/Km, indicating facilitated substrate access to its active site and increased affinity toward substrates with branched chain amino acids in the P1 position. It is suggested that overexpression of LMP7 in the lung is related to increased exposure of the airways to foreign antigens. The possible association between amounts of LMP7 and the activation of the BrAAP component needs further examination.

Topics: Amino Acid Sequence; Animals; Catalysis; Cattle; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Isocoumarins; Liver; Lung; Molecular Sequence Data; Multienzyme Complexes; Pituitary Gland; Proteasome Endopeptidase Complex; Proteins

The serpin mechanism of action may resemble the "standard mechanism" described for small protein inhibitors of serine proteinases. Since these inhibitors are able to bind active site-modified target proteinases, we have investigated the interactions between two serpins and their 3,4-dichloroisocoumarin (DCI)-inactivated target proteinases. alpha 2-Antiplasmin and alpha 1-proteinase inhibitor bound stoichiometrically to DCI-inactivated chymotrypsin (EC 3.4.21.1) and DCI-inactivated human neutrophil elastase, respectively. Similar to active proteinases, the DCI-inactivated proteinases failed to bind complexes between serpins and synthetic reactive site loop peptides. Thus, the abilities of active and inactive proteinases to bind the serpins probably depend on the same structural characteristics. The thermodynamic stability of the alpha 2-antiplasmin-DCI/chymotrypsin and alpha 1-proteinase inhibitor-DCI/human neutrophil elastase complexes was similar to that of virgin serpins. However, in mouse plasma elimination studies the two complexes were removed rapidly from the circulation, suggesting that they have adopted the receptor recognized conformation. Consequently, cleavage of the reactive center peptide bond and formation of an inhibitor-acyl enzyme complex is neither obligatory to serpin-proteinase complex formation nor essential for the conformational change responsible for receptor mediated endocytosis.

Topics: alpha-2-Antiplasmin; Amino Acid Sequence; Animals; Chymotrypsin; Coumarins; Endopeptidases; Enzyme Stability; Humans; Isocoumarins; Leukocyte Elastase; Mice; Molecular Sequence Data; Pancreatic Elastase; Protein Conformation; Receptors, Cell Surface; Serpins; Thermodynamics

Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition

Topics: Amino Acid Sequence; Animals; Calcium; Cattle; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Isocoumarins; Kinetics; Lauric Acids; Magnesium; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Pituitary Gland; Protease Inhibitors; Proteasome Endopeptidase Complex; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Substrate Specificity; Trypsin

The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Cattle; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Endopeptidases; Enzyme Activation; Fatty Acids; Heparin; Immune Sera; Isocoumarins; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Phosphorylation; Pituitary Gland; Proteasome Endopeptidase Complex; Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Substrate Specificity; Trypsin