Alkaline proteases in the midgut of Manduca sexta.

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.