Leonard M. Hjelmeland

Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities, and purification of the dye

Abstract Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.

Sulfobetaine derivatives of bile acids: Nondenaturing surfactants for membrane biochemistry

The syntheses of four new sulfobetaine derivatives of bile salts are presented, along with a general set of criteria for useful detergents in membrane biochemistry. Physical properties including the critical micelle concentration, aggregation number, partial specific volume, critical micellar temperature, uv-vis spectrum, and circular dichroism spectrum are examined for the new compounds. To examine the interaction of this class of compounds with macromolecules, one of these (CHAPS) was further studied. Circular dichroism spectra of apolipoprotein C-III2 were measured in the presence of varying concentrations of CHAPS to determine the effect of this compound on secondary structure. Gel-exclusion chromatography and sedimentation equilibrium studies of cytochrome P-450 in the presence of CHAPS were also performed to establish the ability of this detergent to disaggregate cytochrome P-450 to a monomeric/dimeric state.

The Ah locus, a multigene family necessary for survival in a chemically adverse environment: comparison with the immune system.

Publisher Summary This chapter discusses the drug-metabolizing enzyme systems, presents the Ah system, and provides data for multiple Ah-structural gene products. A cytosolic receptor is regarded as the major product of the Ah-regulatory genes. Sucrose-density gradient analysis following dextran–charcoal treatment is among the most reliable methods for characterizing an Ah receptor. The aryl hydrocarbon hydroxylase (AHH) fluorescent assay is simple and extremely sensitive. This assay, using benzo[α]pyrene as the substrate in vitro, is most commonly used as the biochemical marker for the Ah locus in laboratory animal and human studies. When males and females of four inbred strains were housed together and allowed to breed randomly for 46 to 48 months, the AHH inducibility by 3-methylcholanthrene of weanlings between 18 and 22 generations approximated the distribution found normally for out-bred or random-bred mouse strains. These data indicate the involvement of multiple Ah regulatory genes and a “natural selection” tendency of the induction process to “drift” toward lower intensity in populations having heterogeneous genetic input.

Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents

Abstract In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB14, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7–11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0–4°C, at 200 V in 6-cm gels of 5% T/15% CBis with 0.1 n H2SO4 and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.

A new class of nonionic detergents with a gluconamide polar group

Detergents containing either a cholic acid, a deoxycholic acid, or an octanoic acid-like hydrophobic moiety and a bisgluconamidopropyl polar group were synthesized. Extinction coefficients, partial specific volumes, critical micelle concentrations, and aggregation numbers were determined for each of the detergents. The two bile acid derivatives are capable of solubilizing functional opiate receptor, while the octanoic acid derivative is not.

Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver

A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo[a]pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo[a]pyrene and 4-nitrophenol, and was designated GTM2 .

Induction of microsomal dimethylnitrosamine demethylase by pyrazole

Pyrazole, a potent inhibitor of alcohol dehydrogenase, was found to be a potent inducer of the activity of low Km dimethylnitrosamine demethylase (DMN-d). One injection of pyrazole (200 mg/kg body wt) to weanling Wistar rats changed the microsomal DMN demethylase activity by 1.7, 1.9 and 2.5 times the control values at 6, 12 and 24 hr after the injection respectively. Pyrazole administration reduced arylhydrocarbon hydroxylase (AHH) activity. When animals were injected with pyrazole (200 mg/kg body wt) for 1, 2, 3 or 4 consecutive days, the values for DMN-d activity were 277, 297, 306 and 319% of the control values. The corresponding values for AHH were 91, 67, 57 and 45% for 1, 2, 3 and 4 injections respectively. pyrazole-induced DMN-d activity was NADPH dependent and was inhibited by CO; n-butanol gave a 50% inhibition at a concentration of 2 X 10(-3) M. The corresponding value for metyrapone was 1 X 10(-2) M. Cytochrome P-450 was slightly increased by pyrazole and its CO-complex gave an absorption maximum around 451 nm. When the microsomal proteins were separated using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, a large increase in a band at about 51,000 daltons was found in the liver microsomes of pyrazole-treated animals.

GTP stabilization of adenylate cyclase activated and ADP-ribosylated by choleragen

Abstract Choleragen activates adenylate cyclase in human skin fibroblasts by catalyzing the ADP-ribosylation of the 42,000 and 47,000 dalton guanyl nucleotide-binding regulatory components (G) of adenylate cyclase. The ADP-ribose linkage to 42,000 and 47,000 dalton proteins was stable at 30°C for 1 h with or without GTP, whereas GTP was required to stabilize activity of the G proteins. In human erythrocytes, choleragen catalyzed the ADP-ribosylation of only a 42,000 dalton G. The ADP-ribosyl-protein linkage was stable for 1 h at 30°C whether or not GTP was present, despite a rapid loss of G activity in the absence of GTP. Inactivation of choleragen-activated G in both the human fibroblast and human erythrocyte is, therefore, not secondary to the de-ADP-ribosylation of specifically labeled G subunits.

New Electrophoretic Approaches Applicable to Cell Separation

ABSTRACT To date, separation on the basis of surface net charge differences by electrofocusing has led to loss of cell viability. Potentially, application of the following new approaches to electrofocusing may allow for “charge separation” of viable cells: a) Replacement of Ampholine by ordinary buffers as carrier constituents. b) Replacement of strong acid and base anolyte and catholyte by aminoacids or buffers. Thus, flat pH gradients may be generated and tailored to suit the specific fractionation, either by choice of carrier constituents, or by selective constituent displacement through choice of specific anolytes and catholytes. c) Electrofocusing at high ionic strength. d) Disaggregation by amphoteric detergents capable of maintaining native conformations and biological activities. e) For preparative purposes, zone mobilization in electrofocusing may be achieved by either constituent displacement through change of anolyte, or by “cascade stacking.” Alternatively, material from isoelectric zones can be recovered by steady-state stacking. The present assumption that cell separation by gel electrophoresis, based on differences of size and shape among species, is ruled out by our inability to make gels with large enough pore sizes may be invalid. Potentially, 30–50% Bis-crosslinked polyacrylamide gels, low percent agarose or Pevikon gels and viscous solutions of linear polyacrylamide in an anticonvectant medium seem worth testing on cells 1 with regard to their capability for providing “size fractionation.” Proper choice and design of detergents provides solvents capable of suppressing aggregation while maintaining native structures and functions. Regulated concentration of cells within starting zones can be achieved by use of multiphasic buffer systems of any chosen pH.