A rapid filtration assay for cAMP

The receptor-binding assay for cAMP was improved by using polyethylenimine-treated glass filters. A polyethylenimine-treated glass filter has high protein binding capacity. This high capacity allows an increase in the amount of protein per assay tube and the use of a crude preparation, such as a beef heart extract, as specific binding protein instead of a purified protein, which has been used in the classical filtration assays involving cellulose ester filters. Since the time required for the separation of the protein-cAMP complex and the free nucleotide can be shortened by the use of polyethylenimine-treated filters, the dissociation of the bound ligand during the separation procedure, which is a serious problem with other modified assay methods involving charcoal adsorption, is minimized. Filtration through polyethylenimine-treated glass filters also gives low blanks and prevents the loss of protein or ligand due to breakage of the filters, which is often observed with fragile cellulose ester membranes. In consequence, this simple and rapid filtration assay allows more accurate and reproducible determinations.     

Two simple methods for quantifying low-affinity dye-substrate binding

Binding with low-affinity ligands, such as histological dyes, can be difficult to quantitate owing to the dissociation of bound ligand with washing or the retention of nonspecifically bound ligand because of incomplete washing. The present report describes two simple, rapid methods of discriminating bound from free ligand without the need for washing steps. One method is based on the spectral changes induced in a dye ligand, Congo red, on binding to the "receptor" insulin fibrils. This method discriminates spectrophotometrically between bound and free ligand without requiring any physical separation of the two forms. No radioactive ligands are necessary, and, by using disposable cuvettes, the entire binding assay can be done in a single container without the need for transfers. The second method employs a non-traditional filtration approach that avoids the need for a washing step by measuring the decrease in concentration of the dye ligand in the filtrate rather than by applying the usual approach of measuring the absolute amount of ligand bound to the precipitated "receptor." Both methods show saturation of binding sites and give similar values for the KD and Bmax.     

Cytochrome P-450-dependent 14 alpha-demethylation of lanosterol in Candida albicans.

Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors.     

Effect of the incomplete separation of bound and free ligand on binding measurements

An incomplete separation of free and acceptor-bound ligand causes underestimation of specific binding even though the determinations are corrected with blank or nonspecific binding values. If the failure of the experimental procedure causes contamination of bound ligand with free ligand, Scatchard plots are linear, though their slopes and abscissa intercepts are different from the true ones. On the other hand, if the ligand-acceptor complex is incompletely recovered, Scatchard plots are curvilinear with downward concavity. These problems can be overcome if the separable fractions of free and bound ligand are measured and suitable corrections are applied.The separation of free and receptor-bound ¹²⁵I-labeled human growth hormone by a precipitation method is taken as an example of the procedure.     

Incorporation of P and Growth of Pseudomonad UP-2 on n-Tetracosane

Cultures of the marine pseudomonad UP-2 growing on n-tetracosane contained both free cells and cells bound to the solid hydrocarbon. After separation by filtration through a Whatman no. 1 filter, the numbers of free and bound cells were estimated from the amount of P incorporated into each fraction and the determined value of P incorporation per viable cell in the filtrate (free cells). During the early exponential growth phase, over 80% of the cells were bound to large pieces of n-tetracosane; as the culture approached the stationary phase, the number of bound cells remained constant, whereas free cells continued to accumulate. Pulse-labeling experiments indicated that cells grew both on the surface of the solid and in the aqueous medium. During the growth cycle, a portion of the n-tetracosane which was initially nonfilterable was recovered in the filtrate in a form which was largely cell associated. This cell-associated n-tetracosane was preferentially utilized and could completely account for the observed growth of free cells.     

Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes

A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross- contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.     

Incorporation of 32P and Growth of Pseudomonad UP-2 on n-Tetracosane

Cultures of the marine pseudomonad UP-2 growing on n-tetracosane contained both free cells and cells bound to the solid hydrocarbon. After separation by filtration through a Whatman no. 1 filter, the numbers of free and bound cells were estimated from the amount of ³²P incorporated into each fraction and the determined value of ³²P incorporation per viable cell in the filtrate (free cells). During the early exponential growth phase, over 80% of the cells were bound to large pieces of n-tetracosane; as the culture approached the stationary phase, the number of bound cells remained constant, whereas free cells continued to accumulate. Pulse-labeling experiments indicated that cells grew both on the surface of the solid and in the aqueous medium. During the growth cycle, a portion of the n-tetracosane which was initially nonfilterable was recovered in the filtrate in a form which was largely cell associated. This cell-associated n-tetracosane was preferentially utilized and could completely account for the observed growth of free cells.     

Receptor analysis: An arithmetic correction improves precision and accuracy

Data of receptor analysis by ligand binding experiments should be processed using the formula DCORR = (B1 - B2.F1/F2)/VS.DCORR is an estimate of the concentration of receptor-bound radioligand; B1 and F1 are estimates of bound and free radioligand in assay 1; B2 and F2 are the corresponding values obtained from the parallel assay 2, which contains an additional excess of nonlabeled ligand; VS is the volume of assays 1 and 2 that was submitted to separation. DCORR will be superior to the conventional formula, D = (B1 - B2)/VS, if the radiolabeled receptor-ligand complexes are incompletely separated from nonspecifically bound and free radioligands. DCORR corrects for the systematic underestimation of the specifically bound radioligand implicated in D as well as for random errors due to imprecise pipetting during preparation of the parallel assays. The superiority of DCORR over D is verified by processing the data of androgen receptor analyses using agar gel electrophoresis for separation of bound and free radioligand.     

Adenosine triphosphate-induced rapid calcium release from fragmented sarcoplasmic reticulum

Calcium efflux from skeletal muscle fragmented sarcoplasmic reticulum was studied using a dilution technique and Millipore filtration. In the absence of Mg⁺⁺ and external Ca⁺⁺, addition of lmM adenosine triphosphate to the suspension resulted in an immediate loss of 26–55% of total vesicular calcium. The amount of calcium released was calculated to be sufficient to effect muscle contraction. After separation of the sarcoplasmic reticulum into light, intermediate and heavy vesicles, the light and heavy fractions were found to be only weakly responsive to adenosine triphosphate, whereas the intermediate fraction lost nearly half of its calcium. The significance of these results with respect to excitation-contraction coupling in muscle is discussed.     

Comparing binding of histamine and H2-antagonist with their effects on gastric acid secretion

A microsomal fraction from isolated frog gastric mucosa was used to study the binding of labeled histamine, labeled metiamide (a histamine H2-antagonist), and competition between labeled histamine and unlabeled metiamide. The separation of free from bound ligand was done by gel chromatography. The acid secretion was studied in frog gastric mucosa in vitro by a pH-stat method. The binding data could be interpreted in terms of two independent binding sites for both histamine and metiamide. However, the competition between histamine and metiamide does not support the independence of the sites. Moreover, the dissociation kinetics of labeled metiamide in the presence of unlabeled metiamide is non-monotone and, thus, indicates cooperativity. In the physiological studies, the dependence of the rate of acid secretion on histamine stimulation occurs within very narrow limits, which is the result of characteristics other than related to binding. However, the total amount of acid secreted caused by a pulse of histamine does indicate two sites, of which the high-affinity site is the more effective. Metiamide inhibition of acid secretion can be interpreted as an interaction between high-affinity sites of histamine and metiamide. Overall, studies involving physiological effects provide less precise data than the direct binding studies.     

A simple procedure for the purification of calmodulin bound to membranes; calmodulin bound to the particulate fraction of AH-66 hepatoma ascites cells

We have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.     

A pitfall in the interpretation of data on ligand-protein interaction.

Binding reactions are usually investigated by separation of free and bound labelled ligand. Underestimation of the amount bound leads to inaccurate estimation of the dissociation constant and to wrong conclusions on the validity of the model of binding. The activation of cyclic AMP-dependent protein kinase is treated as an example.     

Effects of proteolytic enzymes on steroid release from rat adrenal zona glomerulosa tissue: Evidence for novel steroid-protein complexes

Trypsin (2mg/ml) added to conventional incubations of rat adrenal capsules (largely glomerulosa) reproducibly increases the amount of free extractable aldosterone (aldo) and 18-hydroxycorticosterone (18-OH-B) in incubation media, but has no effect on capsule cell suspensions formed by collagenase incubation. The effect is abolished by the addition of a trypsin inhibitor, but is still seen in the absence of $\text{de novo}$ steroidogenesis. Qualitatively similar results were obtained with capsule homogenates and high speed supernatant fractions, and chromatography of the high speed supernatant protein fraction on Sephadex G-50 gave a number of minor fractions and one major fraction which yielded free aldo on incubation with trypsin. The results indicate the existence of storage forms of aldo and 18-OH-B which are extremely tightly bound to protein. Such steroid-protein complexes appear to be of an entirely novel kind, and are quite distinct from the familiar receptor type complexes. The findings support previously proposed mechanisms for aldo synthesis and secretion.     

Direct determination of endothelin receptor antagonist levels in plasma using a scintillation proximity assay

An assay using scintillation proximity bead technology has been developed suitable for the quantitation of endothelin (ET) receptor antagonists in preclinical and clinical samples of plasma. The assay measures the competitive inhibition of radiolabelled ET-1 binding to ET(A) receptor membranes bound to wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads in the presence of plasma containing A-127722, a potent orally active, ET(A) selective ET antagonist. The assay requires as little as 50 microl plasma and no extraction procedure is needed. The SPA methodology eliminates the need for the separation of bound from free ligand. Using this method, A-127722 could be directly quantified in rat plasma with a detection limit of 1 ng/ml.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Steroid Receptors in the Rat Hippocampus: A Note to the Methodology of their Binding Assay

Abstract

Mineralocorticoid (MR) and glucocorticoid (GR) receptors in the rat hippocampus are linked to several cognitive functions of the animal and seem to play an important role in the response to various stressors. Their assessment by binding experiments brings about problems associated with their intracellular compartmentalization, and in particular with the separation of the bound and free ligands. Adrenalectomy 24 h before sacrificing is commonly used to clear the circulating adrenal steroids, and to facilitate their dissociation from hippocampal MR and GR. We have successful attempted to use dialysis to these purposes and thus, to avoid a potential surgical stress. Without dialysis, only GR can be measured in the cytosol from intact rats, while the corresponding pellet contains MR as a component of the cell nuclei. The bound ligand fraction was separated by filtration on polyethyleneimine pretreated glass fiber filters as suggested earlier. The method has clear-cut preferences compared to any alternative used up to now. Discrimination between the two receptor types can be optimally achieved in a cross-displacement experiment in which two labeled ligands possessing various affinities to individual receptors (in our case: corticosterone and aldosterone, or their synthetic analogs) are displaced with the two corresponding nonlabelled ligands from their receptors. Computations can be carried out with LIGAND software which yield accurate values of binding parameters.     

High molecular weight complexes of folic acid in mammalian tissues

Twenty-four hours after injection of tritiated folic acid into normal rats, a large amount of labeled folates were found to be associated with the high molecular weight fraction of liver, kidney and intestine. The bound folate was associated with three fractions in the liver cell supernatant having approximate molecular weights of ? 350,000, 150,000 and 25,000 daltons, respectively. A fourth fraction which had an approximate molecular weight of 90,000 daltons was isolated from the liver nuclear fractions. The bound folates associated with these fractions were almost exclusively polyglutamate forms.     

A method for determining kinetic parameters at high enzyme concentrations.

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.     

Study on chemical species of iodine in human liver

The distribution and chemical species of iodine in various subcellular fractions of human liver were studied by using epithermal neutron activation analysis combined with chemical and biochemical separation techniques, such as gradient centrifugation and gel chromatography. It was found that the total iodine content orders in various subcellular fractions is as follows: nuclei > cytosol > mitochondria > lysosome > microsome. In the lysosomal fraction, iodine is mainly bound to macromolecules, whereas in the nuclei and mitochondrial fractions, mainly with lower-molecular-weight organic compounds. In the cytosol fraction, iodine is combined with three proteins, in which iodine is chiefly bound with mid- and high-molecular-weight proteins.     

Biosynthesis of collagen and other proteins on tightly and loosely bound polyribosomes from chick embryos]

Free polyribosomes and polyribosomes bound to endoplasmic membranes were isolated from 10-day-old chick embryos by differential centrifugation. The tightly and loosely bound polyribosomal fractions were isolated from the membrane-bound polyribosomes using 0,5 M KCl. The synthesis of collagen and non-collagen proteins on the polyribosomes were studied in a homologous cell-free system. It was shown that the polyribosomes tightly bound to the membranes possess a lower protein-synthesizing activity as compared to free and loosely bound polyribosomes. The amount of bacterial collagenase-cleaved polypeptides in the protein product synthesized on the polyribosomes tightly and loosely bound to the memranes and on free polyribosomes is 31, 23 and 9%, respectively. The data obtained suggest that the loosely bound polyribosomes are actively involved in collagen synthesis and that this fraction is not a contamination of free polyribosomes in the preparations of totally bound polyribosomes. The role of tightly and loosely bound polyribosomes in the formation of the membrane polyribosomal complex is discussed.