Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate

The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K(i) 1.1 microM, and uncompetitive with respect to shikimate-3-phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate-3-phosphate complex.     

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.     

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.     

A high recovery method for concentrating microgram quantities of protein from large volumes of solution

A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.     

A method for the ligation of DNA following isolation from low melting temperature agarose

A rapid, simple, and reliable method is presented for the isolation and subsequent ligation of DNA from agarose gels. The technique involves the use of low melting temperature agarose, but with the inclusion of bovine serum albumin or gelatin to the ligation reaction.     

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels,exemplified by purification of a retinol-binding protein

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.     

Fast-Transported Glycoproteins and Nonglycosylated Proteins Contain Sulfate

35SO4-labeled fast-transported proteins of bullfrog dorsal root ganglion neurons were separated by two-dimensional gel electrophoresis, and their mobilities were compared to similar species labeled with [3H]mannose or [3H]fucose. Fluorography revealed regions of poorly resolved, high molecular weight material, likely to represent sulfated proteoglycans, as well as many well resolved spots that corresponded in mobility to individual [35S]methionine-labeled fast-transported proteins. The majority of these well resolved spots appeared as "families," previously identified as glycoproteins based on their labeling with sugars. Thus, sulfate can be a contributor to the carbohydrate side-chain charge that underlies microheterogeneity. The most heavily 35SO4-labeled species, however, corresponded to fast-transported proteins that were not labeled by either sugar. The relative acid labilities of 35SO4 associated with individual species cut from the gel confirmed the assignments of these spots as glycoproteins or nonglycoproteins. A group of spots intermediate in their acid lability was also detected, suggesting that some proteins may contain sulfate linked to carbohydrate as well as to amino acid residues.     

Electrophoretic analysis of a chromosomal hybrid zone in the grasshopper Podisma pedestris

We have made an extensive allozyme survey of 21 enzyme and protein loci in populations of the alpine grasshopper Podisma pedestris. This species occurs in two races, differing by a chromosomal fusion which separates the ancestral XO/XX race from a derived neo-XY race. These races also differ in DNA content, and hybrids between them have reduced viability. Electrophoresis reveals that the amount of genetic differentiation between these races is no greater than the variation among populations within each race. Both larger-scale surveys and a detailed survey of an area where the races hybridize, show that the chromosomal change is not correlated with gene frequency changes at any of the 21 loci studied. These findings are consistent with recently developed theory concerning the strength of the barrier to gene flow posed by a hybrid zone with characteristics such as those measured experimentally in Podisma. It is argued that hybrid zones in other species which involve allozymic differences do so because of stronger selection against hybrids rather than through mating isolation.     

The establishment of cell lines from imaginal wing discs of Spodoptera frugiperda and Plodia interpunctella

Continuous cell cultures were established from imaginal wing discs of 2 Lepidoptera, Spodoptera frugiperda and Plodia interpunctella. The S. frugiperda line (IAL-SFD1) grows as multicellular vesicles and responds morphologically and biochemically to the insect hormone, 20-hydroxyecdysone. In contrast, the P. interpunctella cells (IAL-PID2) grow as attached monolayers of small spindle-shaped cells and do not appear to have specific responses to 20-hydroxyecdysone, although growth rates are slowed in these cells upon exposure to the hormone.     

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori

Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.