title: Revisiting rubisco as a protein substrate for insect midgut proteases.
abstract: Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.
authors: Plant-Insect Interactions Group, Department of Botany, Delhi University, Delhi, India.
authors: Usha
authors: U
authors: Bhardwaj
authors: Amit
authors: A
authors: Bhardwaj
authors: Rakesh
authors: R
authors: Kumar
authors: Sadhu
authors: S
authors: Leelavathi
authors: Vanga Siva
authors: VS
authors: Reddy
authors: Sudeshna
authors: S
authors: Mazumdar-Leighton
mesh_terms: D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D019476:Insect Proteins / Q000378:metabolism; D009036:Moths / Q000201:enzymology* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D019476:Insect Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D012273:Ribulose-Bisphosphate Carboxylase; D004229:Dithiothreitol
keywords: midgut serine proteinases; novel protein substrate; protease inhibitors; recombinant RBCL