Heterogeneity of the Basic Pancreatic Inhibitor (Kunitz) in Various Bovine Organs

Fioretti, Evandro; Binotti, Ines; Barra, Donatella; Citro, Gennaro; Ascoli, Franca; Antonini, Eraldo

doi:10.1111/j.1432-1033.1983.tb07110.x

Cited by 36 publications

(1 citation statement)

References 28 publications

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“…Esta metodologia é utilizada para o isolamento de inibidores básicos, resistentes a ácido, a partir de órgãos de animais ruminantes como; boi, carneiro e alce (FIORETTI et al, 1983;FIORETTI et al, 1990;ELEUTERI et al, 1994). Neste protocolo o extrato bruto é clarificado por elemento filtrante associado à centrifugação para remoção de material particulado, células, organelas e "debris" seguido de precipitação por desnaturação ácida para remoção de proteínas indesejáveis (contaminantes) ainda presentes no sobrenadante.…”

Section: Extração De Inibidores Protéicos De Serinoproteases De Pulmãunclassified

Purificação e caracterização da aprotinina obtida de pulmão suíno.

Dias¹

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Section: Extração De Inibidores Protéicos De Serinoproteases De Pulmãunclassified

Purificação e caracterização da aprotinina obtida de pulmão suíno.

Dias¹

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Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

et al. 1997

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The crystal structure of bovine alpha-chymotrypsin (alpha-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 A resolution (R-factor = 0.18). The proteinase:inhibitor complex forms a compact dimer (two alpha-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between alpha-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P1 residue of BPTI, however, does not occupy the alpha-CHT S1 specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217.

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Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting

Rodgers¹,

Melrose²,

Ghosh³

1996

Electrophoresis

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Biotinylated trypsin (bT) was used as a probe on Western blots of 10-20% polyacrylamide gradient Tris-Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S--300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of approximately 6 kDa was also detectable in cartilage extracts after prolonged storage at 4 degrees C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight 'native' cartilage SPI was distinct from ovine alpha1-proteinase inhibitor (alpha1-PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)-PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.

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Heterogeneity of the Basic Pancreatic Inhibitor (Kunitz) in Various Bovine Organs

Cited by 36 publications

References 28 publications

Purificação e caracterização da aprotinina obtida de pulmão suíno.

Purificação e caracterização da aprotinina obtida de pulmão suíno.

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting

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