George G. Berg

Changes in the properties of human erythrocyte membrane protein after solubilization by butanol extraction

1. 1.|Human erythrocyte membrane protein is readily solubilized and freed of most of the membrane lipid after a single extraction of an aqueous suspension of hemoglobin-free ghosts with n-butanol. The technique yields a mean recovery of 83.1% ± 3.8 (S.D., n = 10) of the membrane protein in the butanol-saturated water phase. 2. 2.|The mean amount of lipid present in the water phase after a single extraction is 4.8% ± 3.9 (S.D., n = 6) of the weight of protein. Loss of lipid is symmetrical except that a relatively high proportion of phosphatidyl serine remains. Analysis of the chemical composition of the protein indicates that it is a glycoprotein, which contains hexose, hexosamine, fucose and sialic acid. Carbohydrates account for about 9% of the protein by weight. 3. 3.|Approximately 98% of the protein has a single electrophoretic mobility and appears as a single peak in the void volume after gel column chromatography. Ultracentrifugal analysis, however, demonstrates heterogeneity of the protein. The protein is soluble at neutral pH in salt-containing aqueous media and has an isoelectric point between pH 4.0 and 5.0. 4. 4.|Solubilization of membrane protein by treatment with n-butanol results in a minimal (5%) increase in p-chloromercuriphenylsulfonate titratable SH− groups. 5. 5.|The cation-independent nucleosidetriphosphatase and alkaline monoester phosphydrolase activities of intact stroma were recovered to the extent of 73% to 107% in the solubilized protein. Recoveries of acid phosphohydrolase were 40% at pH 7.0 and 30% at pH 6.0. 6. 6.|The prominent cation activation of both nucleosidetriphosphatase and acid phosphohydrolase activities in stroma was virtually lost on solubilization of the protein. In the case of nucleosidetriphosphatase the concentration of butanol necessary for loss of activation by Mg2+ or by Mg2+, Na+ and K+ was sufficiently high (above 500 mM) to cause disintegration of structure.

Enzyme histochemical studies of human gastric and jejunal biopsy specimens in normal and disease states

SummaryThe alkaline phosphatase activity of 28 gastric and 32 jejunal biopsy specimens examined histochemically revealed the following:(1)absence of alkaline phosphatase activity in normal gastric mucosa and in mucosa with varying degrees of gastritis unassociated with intestinal metaplasia;(2)marked alkaline phosphatase activity in the brush border region of the metaplastic cells in 3 patients with gastritis associated with intestinal metaplasia; and(3)no significant difference in alkaline phosphatase activity between the jejunal biopsy specimens of normal patients and those with primary malabsorption syndrome. The trimetaphosphatase activity of 24 gastric and 32 jejunal biopsy specimens examined histochemically showed:(1)no significant difference in trimetaphosphatase activity between stomach biopsy specimens with normal histology and those from patients with varying degrees of gastritis; and(2)no significant change in trimetaphosphatase activity between the jejunal biopsy specimens of normal patients and those with primary malabsorption syndrome;(3)the variability of trimetaphosphatase activity in the human stomach and jejunum does not allow differentiation between normal and disease states but may reflect transient differences in local activity between different areas selected for biopsy.

Binding of Mercurials to Membrane Suspensions and Undenatured Proteins

Binding capacities of membrane suspensions and dissolved compounds for mercurials were titrated by a new potentiometric method. Critical steps included a silver electrode of new design, the use of L-cysteine as a thiol buffer, a nitrogen atmosphere, and pretreatment of samples with equimolar mercurial and cysteine. Titrations had a sharp endpoint, accurate +/- 26 nmole methylmercury or +/- 8 nmole mercuric salt. Measurements of binding capacity of bovine serum albumin averaged 93% of the titer predicted for one SH group per molecule; those of human hemoglobin yielded 86-91% of the titer predicted for two SH groups per molecule. Yields dropped with exposure of protein solutions or membrane suspensions to atmospheric oxygen. Brain microsomes had significantly higher binding capacities (per milligram of protein) than red blood cell ghosts. The ratio of endpoint titers of CH3HgCl to HgCl2 averaged 2:1 in assays of cysteine, proteins, and membranes, showing that the assay was free of denaturation artifacts and protein-protein interference. Solutions of EDTA showed measurable binding of Hg2+ but not of CH3Hg+. Satisfactory titrations were also obtained with N-ethylmaleimide.

The Risks of Measuring Risk

The Thirteenth Rochester International Conference on Environmental Toxicity examined both the logical soundness of the inference of risk and the validity of the experimental evidence of damage. Examples were drawn from current research by the participants on environmental hazards of toxic chemicals and ionizing radiation. Contributions by twenty-two researchers were grouped under six headings: — Statistical Inference of Risks — Risks of Prenatal Exposures — Risks of Contaminated Air and Drinking Water — Comparative Risks of Energy Sources — Genetic and Cancer Hazards — Extrapolation to Low Doses

Chemical Protection Against Lethal Effects of Nitrogen Mustard in Rats.

Summary 1. A dose-mortality curve for nitrogen mustard (HN2) given intraperitoneally to 150 Wistar rats gave an LD50 of 1.9 ± S.E. = 0.02 mg of HN2 per kg body weight. Mean survival times for groups of rats killed by moderate doses of HN2 (LD10 through 2 × LD50) were of the order of 4 days. Acute toxic doses (above 7 × LD50) killed in 2 days or less. 2. Methyl and propyl paraben mixed into the saline solution used for injecting sub-lethal doses of HN2 had no effect upon either survival time or mortality of the rats. These additives apparently have no adverse effect on the action of HN2 in clinically acceptable doses. 3. A 600 mg% diet of parabens fed to rats receiving sublethal doses of HN2 increased survival time, but did not reduce mortality. Survival time of rats injected with acute toxic doses of HN2 was increased as much as 1 1/2 days by parabens dissolved in the saline vehicle, with no change of mortality. 4. Pre-feeding of a 5% succinylsulfathiazole diet prolonged survival time of rats treated with HN2 consistently and significantly. 5. Tripolyphosphate administered by various routes had no protective effect upon survival time or mortality rate of rats injected with HN2. 6. The mechanism of action of the drugs is discussed.