Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.
We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.
Underlying the risk management of
pesticides to protect human health
and to facilitate trade among nations are sound scientific data on
the levels of compliance with standards set by governments and internationally
from monitoring of the levels of pesticides in foods. Although glyphosate
is among the universally used pesticides in the world, monitoring
has been hampered by the analytical difficulties in dealing with this
highly polar compound. Starting in 2015, using liquid chromatography/tandem
mass spectrometry (LC-MS/MS) that permits accurate and reproducible
determination of glyphosate, the prevalence, concentrations, and compliance
rates were determined. In this work, the glyphosate residues contents
of 7955 samples of fresh fruits and vegetables, milled grain products,
pulse products, and finished foods collected from April 2015 to March
2017 in the Canadian retail market are reported. A total of 3366 samples
(42.3%) contained detectable glyphosate residues. The compliance rate
with Canadian regulations was 99.4%. There were 46 noncompliant samples.
Health Canada determined that there was no long-term health risk to
Canadian consumers from exposure to the levels of glyphosate found
in the samples of a variety of foods surveyed. The high level of compliance
(99.4% of samples with the Canadian regulatory limits) and the lack
of a health risk for noncompliant samples indicate that, with respect
to glyphosates, the food available for sale in Canada is safe.
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