Persistent Link:
http://hdl.handle.net/10150/186860
Title:
Alkaline proteases in the midgut of Manduca sexta.
Author:
Peterson, Ann Michele.
Issue Date:
1994
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The chymotrypsin and trypsin-like activities from the Manduca sexta midgut were purified, partially characterized and the cDNAs encoding the enzymes were cloned. A putative elastase cDNA was also cloned and the sequence is reported. The purified trypsin and chymotrypsin are maximally active at alkaline pH (10.5-11.0 and 10.5 respectively). Kinetic studies of the chymotrypsin reveal that the K(m) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide varies only slightly between pH 7.5-11.5 and the Dixon plot shows a kinetically significant pKₐ at 9.2. The specificity of the purified chymotrypsin is the hydrolysis of the peptide bond on the carboxyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, threonine and glycine. The chymotrypsin is inhibited by TPCK, PMSF, chymostatin and DFP. Three different trypsin cDNAs, two chymotrypsin cDNAs and an elastase cDNA were isolated that encode preproenzymes of 256, 292 and 291 amino acids, respectively. All the encoded activation peptides contain an arginine residue immediately preceding the amino-terminal residue of the mature enzymes which suggests tryptic activation of the zymogens. The encoded trypsins and chymotrypsin contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the specificity pockets. The encoded elastase contains the residues which define the specificity pocket; however, the "catalytic triad" consists of Asp, His and Thr. All the cDNAs for the midgut enzymes encode a large number of arginines which may stabilize the enzymes, by remaining protonated, in the alkaline midgut of M. sexta. Sequence comparisons show that all sequences are most similar to other invertebrate and vertebrate serine proteases. Northern analysis localizes the trypsin mRNA to the middle and posterior third of the midgut, the chymotrypsin mRNA to the anterior and middle third of the midgut and the elastase mRNA to the middle third of the midgut.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Biochemistry; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Wells, Michael A.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleAlkaline proteases in the midgut of Manduca sexta.en_US
dc.creatorPeterson, Ann Michele.en_US
dc.contributor.authorPeterson, Ann Michele.en_US
dc.date.issued1994en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe chymotrypsin and trypsin-like activities from the Manduca sexta midgut were purified, partially characterized and the cDNAs encoding the enzymes were cloned. A putative elastase cDNA was also cloned and the sequence is reported. The purified trypsin and chymotrypsin are maximally active at alkaline pH (10.5-11.0 and 10.5 respectively). Kinetic studies of the chymotrypsin reveal that the K(m) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide varies only slightly between pH 7.5-11.5 and the Dixon plot shows a kinetically significant pKₐ at 9.2. The specificity of the purified chymotrypsin is the hydrolysis of the peptide bond on the carboxyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, threonine and glycine. The chymotrypsin is inhibited by TPCK, PMSF, chymostatin and DFP. Three different trypsin cDNAs, two chymotrypsin cDNAs and an elastase cDNA were isolated that encode preproenzymes of 256, 292 and 291 amino acids, respectively. All the encoded activation peptides contain an arginine residue immediately preceding the amino-terminal residue of the mature enzymes which suggests tryptic activation of the zymogens. The encoded trypsins and chymotrypsin contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the specificity pockets. The encoded elastase contains the residues which define the specificity pocket; however, the "catalytic triad" consists of Asp, His and Thr. All the cDNAs for the midgut enzymes encode a large number of arginines which may stabilize the enzymes, by remaining protonated, in the alkaline midgut of M. sexta. Sequence comparisons show that all sequences are most similar to other invertebrate and vertebrate serine proteases. Northern analysis localizes the trypsin mRNA to the middle and posterior third of the midgut, the chymotrypsin mRNA to the anterior and middle third of the midgut and the elastase mRNA to the middle third of the midgut.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairWells, Michael A.en_US
dc.contributor.committeememberLaw, John H.en_US
dc.contributor.committeememberCusanovich, Michael A.en_US
dc.contributor.committeememberTollin, Gordonen_US
dc.contributor.committeememberGoll, Darrel E.en_US
dc.identifier.proquest9506991en_US
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