The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.     

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Seasonal carbon isotope discrimination in a grassland community

Summary Grassland communities of arid western North America are often characterized by a seasonal increase in ambient temperature and evaporative demand and a corresponding decline in soil moisture availability. As the environment changes, particular species could respond differently, which should be reflected in a number of physiological processes. Carbon isotope discrimination varies during photosynthetic activity as a function of both stomatal aperture and the biochemistry of the fixation process, and provides an integrated measure of plant response to seasonal changes in the environment. We measured the seasonal course of carbon isotope discrimination in 42 grassland species to evaluate changes in gas exchange processes in response to these varying environmental factors. The seasonal courses were then used to identify community-wide patterns associated with life form, with phenology and with differences between grasses and forbs. Significant differences were detected in the following comparisons: (1) Carbon isotope discrimination decreased throughout the growing season; (2) perennial species discriminated less than annual species; (3) grasses discriminated less than forbs; and (4) early flowering species discriminated more than the later flowering ones. These comparisons suggested that (1) species active only during the initial, less stressful months of the growing season used water less efficiently, and (2) that physiological responses increasing the ratio of carbon fixed to water lost were common in these grassland species, and were correlated with the increase in evaporative demand and the decrease in soil moisture.     

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

Requirements for the translocation of diphtheria toxin fragment A across lipid membranes

The translocation of the enzymatic moiety of diphtheria toxin, fragment A, across the membranes of pure lipid vesicles was demonstrated. A new assay, which employed vesicles made to contain radiolabeled NAD and elongation factor-2, was used to measure the appearance of the enzymatic activity of the A fragment in the vesicles. When the vesicles were exposed to a low-pH medium in the presence of diphtheria toxin, small molecules, such as NAD, escaped into the extravesicular medium, whereas large molecules mostly remained inside the vesicles. The vesicle-entrapped elongation factor-2 became ADP-ribosylated, indicating the entry of fragment A into the vesicle. The translocation of the A fragment depended upon the pH of the medium, being negligible at pH greater than 7.0 and maximal at pH 4.5. The entire toxin molecule was needed for function; neither the A fragment nor the B fragment alone was able to translocate itself across and react with the sequestered substrates. After exposure of the toxin to low pH, the entry of the A fragment was rapid, being virtually complete within 2-3 min at pH 5.5, and within 1 min at pH 4.7. Translocation occurred in the absence of any protein in the vesicle membrane. These results are consistent with the notion that the diphtheria toxin molecule enters the cytoplasm of a cell by escaping from an acidic compartment such as an endocytic vesicle.