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The objective is to study a novel protease P isolated from the digestive tract of an Amazonian insect. This protease can exist into two forms Pi and Pa which have identical amino acid sequences (both of 80 kDa). However, only Pa shows proteolytic activity. To better understand the activation mode of Pi (inactive form) in Pa (active form), the following experiment was done using DIPF. DIPF (diisopropylphosphofluoridate) is a well-known irreversible inhibitor of serine proteases. It reacts with the catalytic serine residue of the active site of proteases as shown below: Enzyme -CH₂OH + CH(CH3)2 O FIP=0 CH(CH3)2 Diisopropylphospho- fluoridate (DIPF) Enzyme CH(CH3)2 O -CH₂-O-P=O O CH(CH3)2 DIP-Enzyme + HF Both proteases Pa and P₁ were incubated with 32P-DIPF for 30 min at 37°C, and then dialysed to remove excess of unreacted radiolabelled reagent. The two proteases were then analyzed in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without 2-mercaptoethanol. The gel shown below was stained with Coomassie blue (Figure A). A) Piste 1 Piste 2 Piste 3 Piste 4 Piste 5 B) Piste 1 Piste 2 Piste 3 Piste 4 Piste 5 Molecular weight markers in lane 1 are (in Da): ß-galactosidase 120 000; bovine serum albumin 70 000; ovalbumin 42 000; trypsin 25 000; myoglobin 16 000; peptone 9 000. The protein P₁ is loaded in lanes 2 and 3, without and with 2- mercaptoethanol, respectively. The protein Pa is loaded in lanes 4 and 5, without and with 2-mercaptoethanol, respectively. Interpret SDS-PAGE results and what does this tell you about the activation process of the protein P? An autoradiography of the gel (using a photosensitive film) has been performed to identify which protein bands have incorporated the radioactive reagent. This yielded the result presented in Figure B. To refine your conclusions on the mode of activation of protein P, complete your previous analysis of Figure A by studying the autoradiography results in Figure B.

The objective is to study a novel protease P isolated from the digestive tract of an Amazonian insect. This protease can exist into two forms Pi and Pa which have identical amino acid sequences (both of 80 kDa). However, only Pa shows proteolytic activity. To better understand the activation mode of Pi (inactive form) in Pa (active form), the following experiment was done using DIPF. DIPF (diisopropylphosphofluoridate) is a well-known irreversible inhibitor of serine proteases. It reacts with the catalytic serine residue of the active site of proteases as shown below: Enzyme -CH₂OH + CH(CH3)2 O FIP=0 CH(CH3)2 Diisopropylphospho- fluoridate (DIPF) Enzyme CH(CH3)2 O -CH₂-O-P=O O CH(CH3)2 DIP-Enzyme + HF Both proteases Pa and P₁ were incubated with 32P-DIPF for 30 min at 37°C, and then dialysed to remove excess of unreacted radiolabelled reagent. The two proteases were then analyzed in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without 2-mercaptoethanol. The gel shown below was stained with Coomassie blue (Figure A). A) Piste 1 Piste 2 Piste 3 Piste 4 Piste 5 B) Piste 1 Piste 2 Piste 3 Piste 4 Piste 5 Molecular weight markers in lane 1 are (in Da): ß-galactosidase 120 000; bovine serum albumin 70 000; ovalbumin 42 000; trypsin 25 000; myoglobin 16 000; peptone 9 000. The protein P₁ is loaded in lanes 2 and 3, without and with 2- mercaptoethanol, respectively. The protein Pa is loaded in lanes 4 and 5, without and with 2-mercaptoethanol, respectively. Interpret SDS-PAGE results and what does this tell you about the activation process of the protein P? An autoradiography of the gel (using a photosensitive film) has been performed to identify which protein bands have incorporated the radioactive reagent. This yielded the result presented in Figure B. To refine your conclusions on the mode of activation of protein P, complete your previous analysis of Figure A by studying the autoradiography results in Figure B.

Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
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The objective is to study a novel protease P isolated from the digestive tract of an Amazonian insect. This protease can exist into two forms Pi and Pa which have identical amino acid sequences (both of 80 kDa). However, only Pa shows proteolytic activity. To better understand the activation mode of Pi (inactive form) in Pa (active form), the following experiment was done using DIPF. DIPF (diisopropylphosphofluoridate) is a well-known irreversible inhibitor of serine proteases. It reacts with the catalytic serine residue of the active site of proteases as shown below: Enzyme -CH₂OH + CH(CH3)2 O F-P=0 O CH(CH3)2 Diisopropylphospho- fluoridate (DIPF) Enzyme -CH,—O CH(CH3)2 O <=0 O CH(CH3)2 DIP-Enzyme Both proteases Pa and P₁ were incubated with 32P-DIPF for 30 min at 37°C, and then dialysed to remove excess of unreacted radiolabelled reagent. The two proteases were then analyzed in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without 2-mercaptoethanol. The gel shown below was stained with Coomassie blue (Figure A). A) Piste 1 Piste 2 Piste 3 Piste 4 Piste 5 B) Piste 1 Piste 2 Piste 3 Piste 4 + HF Piste 5 Molecular weight markers in lane 1 are (in Da): ß-galactosidase 120 000; bovine serum albumin 70 000; ovalbumin 42 000; trypsin 25 000; myoglobin 16 000; peptone 9 000. The protein P₁ is loaded in lanes 2 and 3, without and with 2- mercaptoethanol, respectively. The protein Pa is loaded in lanes 4 and 5, without and with 2-mercaptoethanol, respectively. Interpret SDS-PAGE results and what does this tell you about the activation process of the protein P? An autoradiography of the gel (using a photosensitive film) has been performed to identify which protein bands have incorporated the radioactive reagent. This yielded the result presented in Figure B. To refine your conclusions on the mode of activation of protein P, complete your previous analysis of Figure A by studying the autoradiography results in Figure B.
Transcribed Image Text:The objective is to study a novel protease P isolated from the digestive tract of an Amazonian insect. This protease can exist into two forms Pi and Pa which have identical amino acid sequences (both of 80 kDa). However, only Pa shows proteolytic activity. To better understand the activation mode of Pi (inactive form) in Pa (active form), the following experiment was done using DIPF. DIPF (diisopropylphosphofluoridate) is a well-known irreversible inhibitor of serine proteases. It reacts with the catalytic serine residue of the active site of proteases as shown below: Enzyme -CH₂OH + CH(CH3)2 O F-P=0 O CH(CH3)2 Diisopropylphospho- fluoridate (DIPF) Enzyme -CH,—O CH(CH3)2 O <=0 O CH(CH3)2 DIP-Enzyme Both proteases Pa and P₁ were incubated with 32P-DIPF for 30 min at 37°C, and then dialysed to remove excess of unreacted radiolabelled reagent. The two proteases were then analyzed in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without 2-mercaptoethanol. The gel shown below was stained with Coomassie blue (Figure A). A) Piste 1 Piste 2 Piste 3 Piste 4 Piste 5 B) Piste 1 Piste 2 Piste 3 Piste 4 + HF Piste 5 Molecular weight markers in lane 1 are (in Da): ß-galactosidase 120 000; bovine serum albumin 70 000; ovalbumin 42 000; trypsin 25 000; myoglobin 16 000; peptone 9 000. The protein P₁ is loaded in lanes 2 and 3, without and with 2- mercaptoethanol, respectively. The protein Pa is loaded in lanes 4 and 5, without and with 2-mercaptoethanol, respectively. Interpret SDS-PAGE results and what does this tell you about the activation process of the protein P? An autoradiography of the gel (using a photosensitive film) has been performed to identify which protein bands have incorporated the radioactive reagent. This yielded the result presented in Figure B. To refine your conclusions on the mode of activation of protein P, complete your previous analysis of Figure A by studying the autoradiography results in Figure B.
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