Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation.

A screen of a Mamestra configurata (bertha armyworm) midgut cDNA library identified three types of cDNA clones that resemble the Manduca sexta serpin-1 gene family. Two serpins, 1b and 1c, possess a common conserved serpin amino terminal scaffold domain but bear no similarity to any members of the M. sexta gene family within the reactive centre loop. These serpins differ from one another by only two amino acids in the reactive centre loop (S(363)-->P) and serpin signature (M(369)-->T) regions. The other member, denoted serpin-1a, is closely related to the M. sexta serpin-1Z. M. configurata serpins as a group were expressed in all insect developmental stages including eggs, larvae and adult moths. Within larvae, serpin gene expression was restricted to the early to middle instar developmental phase and mainly in the fat body and hemocytes. Stress imposed by starvation strongly induced expression in fat body and to a lesser degree in alimentary organs, nervous system and Malphigian tubules. Conversely, starvation decreased expression in hemocytes. Wounding or inoculation with bacteria did not induce serpin gene transcription but did lead to the formation of higher and lower molecular weight forms, presumably serpin-protease complexes and resultant truncated serpin, respectively. Two dimensional PAGE and western blotting analysis revealed at least 12 distinct serpins consisting primarily of neutral, but also highly acidic and basic isoforms, as well as additional high and low molecular weight immuno-reactive species. Serpins-1b/1c are the more prominent serpin isoforms and are expressed predominantly in the fat body and subsequently exported to the hemolymph as revealed by western blotting and immunolocalization. The serpin-1b/1c isoform was found only as the fully glycosylated species within the hemolymph. Hemolymph protease activity was comprised mostly of serine proteases whose overall activity increased dramatically at the onset of the molt concomitant with a sharp decline in serpin gene expression.

An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.