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Sequence of three cDNAs encoding an alkaline midgut trypsin from Manduca sexta
A M Peterson1, C V Barillas-Mury, M A Wells
1Department of Biochemistry, University of Arizona, Tucson 85721.
Insect Biochemistry and Molecular Biology
|May 1, 1994
Summary
Researchers cloned three midgut trypsin cDNAs from Manduca sexta, revealing enzymes with alkaline pH optima. These insect trypsins share conserved catalytic residues with other serine proteases but exhibit unique cysteine and arginine content potentially aiding function in the alkaline insect gut.
Area of Science:
- Biochemistry
- Molecular Biology
- Insect Physiology
Background:
- Trypsin is a key digestive enzyme in many organisms.
- Understanding insect trypsin is crucial for insect biology and pest control.
- The midgut environment of Manduca sexta is alkaline, suggesting specific adaptations for digestive enzymes.
Purpose of the Study:
- To clone and characterize trypsin cDNAs from the midgut of Manduca sexta.
- To investigate the structural and functional properties of Manduca sexta trypsin.
- To understand the role of trypsin in the alkaline digestive system of this insect.
Main Methods:
- Purification of trypsin from Manduca sexta midgut.
- Generation of a DNA probe using polymerase chain reaction (PCR) with degenerate primers.
- Screening of a cDNA library and isolation of three distinct trypsin cDNAs.
- Northern analysis to determine mRNA localization.
Main Results:
- Three trypsin cDNAs were isolated, each encoding a preproenzyme.
- The mature trypsins consist of 232 amino acids and contain conserved catalytic residues (Asp, His, Ser).
- Two of the three trypsins possess an unusual number of cysteines, and all encode a high number of arginines, potentially for alkaline stability.
Conclusions:
- Manduca sexta midgut trypsin functions optimally at an alkaline pH (10.5).
- The cloned trypsins are homologous to other serine proteases but have unique features.
- High arginine content may confer stability in the alkaline midgut environment, facilitating digestion.