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Biomedical subjects

F M Finn

Publications and source records attributed to F M Finn.

At least 37 records · Page 2Linked to original sources
PubMed1984

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

The ligand N alpha, B1-(6-biotinylamido)hexanoyl-insulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, (iii) wheat germ agglutinin purification, and (iv) insulin-affinity chromatography. NaDodSO4/PAGE of the purified 125I-labeled receptor under nonreducing conditions showed the presence of a major component with an approximate molecular weight of 350,000 and a minor component with a molecular weight of approximately equal to 166,000. Based on the assumption that the degree of labeling is comparable in both components, the material corresponding to the Mr 350,000 peak represents approximately equal to 94% of the receptor preparation as determined by scanning the autoradiograms. The specific insulin binding capacity of the preparation is 18 +/- 6 micrograms of 125I-labeled insulin per mg of protein as determined by the polyethylene glycol assay and analyzed by Scatchard plot. Insulin binding activity was stable at 4 degrees C and pH 7.6 for at least 12 weeks but was destroyed by freezing and thawing. The availability of highly purified receptor afforded the opportunity to explore its precipitability by polyethylene glycol under assay conditions. Whereas trichloroacetic acid precipitated 95% of the 125I-labeled receptor, polyethylene glycol precipitated only 30%. If the specific activity of the receptor is corrected for incomplete precipitability by polyethylene glycol, the apparent specific binding would be 3.5 +/- 1.2 mol of insulin per mol of receptor. These results are in disagreement with the current receptor model, which postulates that 1 mol of receptor (Mr, 350,000) binds 2 mol of insulin. Clearly, the problems associated with the method available for determining insulin binding are sufficiently serious to preclude their use in determining receptor valence.

Avidin↗
PubMed1982

Insulin receptor isolation studies.

The observation that N alpha,B1-biotinylinsulin binds firmly to resins in which succinoylavidin is covalently attached to AH Sepharose 4B and can be retrieved by exposure of the resins to 20 mM biotin provided the basis for the present investigations. Solubilized, partially purified insulin receptor from human placenta binds to affinity resins in which N alpha,B1-biotinylinsulin is noncovalently attached to AH Sepharose 4B-immobilized-succinolylavidin. Exposure of the receptor loaded resin to 20 mM biotin results in liberation of a high molecular weight material containing bound 125I-biotinylinsulin, which precipitates with polyethyleneglycol and cross reacts with human insulin receptor antibodies. The technique is biospecific and appears to be applicable to the purification of insulin receptors on a preparative scale. Crude solubilized insulin receptor from human placenta is contaminated with "insulinase" which is inhibited by N-ethylmaleimide. HPLC provides a tool to assess "insulinase" activity that is more sensitive than the TCA precipitation method.

Chromatography, High Pressure Liquid↗
PubMed1981

Isolation and characterization of two adenosine 3',5'-monophosphate-dependent protein kinases from bovine adrenal cortex.

Bovine adrenocortical cytosol contains two cAMP-dependent protein kinases separable by diethylamino-ethyl-cellulose chromatography. The kinases exhibit behavior characteristic of type I and type II enzymes i.e. the enzyme eluting at 80 mM NaCl is activated by NaCl and histone, whereas the enzyme eluting at 140 mM NaCl is not. In addition, a third cAMP-binding protein eluting from DEAE at 120 mM NaCl has been isolated and tentatively identified as type I regulatory subunit. A method is described for the isolation of the catalytic and regulatory subunits of both enzymes from the same starting material using cGMP to dissociate the enzyme subunits and elute the regulatory subunits from N6-aminoethyl-cAMP-Sepharose 4B. The isolated proteins were homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis. Using this technique, molecular weight values of 44,000 for the catalytic subunits, 51,000 for the cAMP-binding protein and type I regulatory subunit, and 56,000 for the type II regulatory subunit were obtained. Molecular weights obtained by sucrose density gradient centrifugation on the cGMP-dissociated holoenzymes were 95,000 and 101,000 for regulatory subunits from type I and II enzymes and 34,000 and 33,000 for their respective catalytic subunits. The apparent Km and Vmax values for catalytic subunits I and II were similar when histone, casein, or ATP was used as substrate. The Vmax for protamine phosphorylation was 3-fold higher with catalytic subunit II. Phosvitin was not a substrate for either subunit. The cAMP-binding protein and regulatory subunit I were able to recombine with, i.e. inhibit, either catalytic subunit. At a molar ratio of 4:1, inhibition was total. The type II regulatory subunit was considerably less effective. Preference for a particular catalytic subunit was not evident in the case of inhibition by either regulatory subunit or the cAMP-binding protein.

Adrenal Cortex↗
PubMed1981

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes.

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.

Adenylyl Cyclases↗
PubMed1980

Hormone-receptor studies with avidin and biotinylinsulin-avidin complexes.

Avidin can be labeled to high specific radioactivity by introducing 3-(p-hydroxyphenyl)-propionyl groups into the molecule (pHPP-avidin). 125I-pHPP-avidin binds avidly to rat liver plasma membranes and is not displaced by unlabeled pHPP-avidin. Nonspecific binding of 125I-pHPP-avidin can be substantially reduced by succinoylation of pHPP-avidin with succinic anhydride (SpHPP-avidin). Spectral changes ensuing when the dye 4-hydroxyazobenzene-2'-carboxylic acid binds to avidin cannot be used to assess the binding characteristics of the modified avidins since the absorption coefficients of the complexes are markedly different; however, the modified molecules bind theoretical amounts of [14C]biotin. Biotinylinsulin and biotinylinsulinSpHPP-avidin complexes compete with 125I-insulin for binding to receptor sites on rat liver plasma membranes. Biotinylinsulin complexes with unmodified avidin display anomalous binding behavior attributable to formation of membrane aggregates. In light of this finding, results obtained using unmodified avidin must be interpreted with caution. Biotinylinsulin125I-SpHPP-avidin binds specifically and saturably to rat liver plasma membranes. The biotinylhormoneSpHPP-avidin technique has potential for labeling peptide hormones and other compounds that cannot be iodinated by conventional procedures.

Amino Acid Sequence↗
PubMed1980

Iminobiotin affinity columns and their application to retrieval of streptavidin.

A method is described for the retrieval of streptavidin from the culture broth of Streptomyces avidinii. The key step in this procedure is the adsorption of streptavidin from culture concentrates to an affinity column in which iminobiotin is attached to AH-Sepharose 4B. This column binds streptavbidin at pH 11 and releases the protein at pH 4. The recovery of streptavidin is practically quantitative. The pH dependence of the iminobiotin-avidin affinity, discovered by Green [Green, N. M. (1966) Biochem. J. 101, 774-779], has thus found practical application. The streptavidin bound 4.07 +/- 0.02 mol of [14C]biotin per mol and was essentially homogeneous as judged by disc and slab gel electrophoresis. Streptavidin was extensively succinoylated without loss of biotin-binding capacity. The observations that 125I-labeled streptavidin and 125I-labeled succinoylstreptavidin are retained by iminobiotin-AH-Sepharose 4B columns at pH 7.5 and are eluted at pH 4.0 provides a convenient purification method for these iodinated proteins. The technique employed for the retrieval of streptavidin is generally applicable to the isolation of iminobiotinylated molecules.

Bacterial Proteins↗
PubMed1980

Ascorbic acid transport by isolated bovine adrenal cortical cells.

Viable, ACTH-sensitive, bovine adrenal cortical cells have the ability to accumulate [1-14C]ascorbic acid. Transport of the vitamin displays saturation kinetics (Km = 16.6 microM), requires Ca2+, and is inhibited maximally by ACTH at the same concentrations of the hormone necessary for maximal stimulation of steroidogenesis. Despite the transport of quantities of radioactive ascorbate amounting to approximately 25% of the total intracellular pool, no change in the ascorbate concentration in the cells can be detected by chemical methods. Transport of the vitamin is apparently a membrane carrier-mediated exchange process, since although intracellular ascorbate concentrations are 60- to 90-fold higher than those in the incubation medium, accumulated label can only be displaced when the cells are incubated in the presence of unlabeled ascorbic acid or metabolic inhibitors. Furthermore, ACTH fails to deplete ascorbic acid from [1-14C]ascorbate-loaded cells. It is hypothesized that the vitamin exists in two pools only one of which can be depleted by ACTH. This ACTH-depletable pool is no longer present in the isolated cell. In fact, as much as 66% of the ascorbic acid is lost from rat adrenals incubated for 15 min post adrenalectomy. The remaining pool of adrenal ascorbate is not depleted by ACTH and is also maintained against a concentration gradient.

Adrenal Cortex↗
PubMed1977

Biotinylinsulins as potential tools for receptor studies.

The preparation of affinity columns that contain insulin attached to Sepharose in a targeted manner by way of biotin-avidin noncovalent bonds is described. Insulin was acylated selectively at the amino terminus of the B chain with the N-hydroxysuccinimido ester of biotin to form N(alpha,B1)-biotinylinsulin. The ability of this modified insulin to stimulate rat epididymal adipocytes was (mean +/- SD) 94 +/- 9.6% (P, 0.05) that of the control insulin. N(alpha,B1)-Biotinylinsulin displaced 4-hydroxyazobenzene-2'-carboxylic acid from avidin, demonstrating affinity for this protein. The formation of the N(alpha,B1)-biotinylinsulin-avidin complex was visualized by cellulose acetate electrophoresis at pH 4. N(alpha,B1)-Biotinylinsulin combined with avidin attached to Sepharose to form affinity columns in which the hormone was attached to the support by strong noncovalent bonds. The determination of the loading of avidin-Sepharose columns with biotinylinsulin was greatly facilitated by the attached biotin which provided a marker whose concentration could be assessed accurately by titration with avidin. Biotinylinsulin attached to avidin-Sepharose beads retained the ability to stimulate rat epididymal adipocytes. The activity of several samples of these beads was about 15% that of free biotinylinsulin, based on the amount of biotinylinsulin anchored to the support. The advantages of biotinylated hormones for the targeted attachment of hormones to solid supports are discussed.

Adipose Tissue↗
PubMed1976

Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells.

The ability of three analogs of ACTH1-24 ([Gln5, Phe9] ACTH1-24, [Gln5, Ala9[Acth1-24, and [Gln5, Lys8, Phe9[ ACTH1-24) embodying tryptophan substitutions to activate the adenylate cyclase system of a bovine adrenal plasma membrane preparation was compared to the effect of the analogs on adenosine 3':5'-monophosphate (cyclic AMP) accumulation and steroidogenesis in viable bovine adrenocortical cells. The results were not comparable. Whereas the analogs antagonized the ACTH1-24-activated membrane cyclase they stimulated cyclic AMP accumulation as well as steroid production of the cells. None of the analogs inhibited steroidogenesis of ACTH1-24-stimulated cells, but two of them, at very high dose levels, inhibited cyclic AMP production. The ability of the analogs to stimulate steroidogenesis of the adrenal cells half-maximally decreased in the order tryptophan greater than phenylalanine greater than alanine, indicating that the aromaticity of the indole ring of tryptophan is necessary for maximal interaction between hormone and receptor. Both the absolute and relative steroidogenic potencies were the same for several analogs when assayed with rat adrenal cells. Although only a small fraction of the cell's potential to produce cyclic AMP was necessary to induce maximum steroid production, the relative activities of a series of analogs were the same for steroidogenesis as for cyclic AMP accumulation. Furthermore, the concentration of cyclic AMP necessary for full steroidogenesis was practically identical for a series of peptides that differed widely in potency. These findings support the postulate that cyclic AMP accumulation and steroidogenesis in adrenocortical cells are coupled processes. The differential behavior of bovine adrenal plasma membranes and bovine adrenocortical cells toward ACTH analogs indicates that structure-function studies using cyclase assays may not reflect events that take place in the intact adrenal or in cell preparations derived therefrom.

Adenylyl Cyclases↗
PubMed1975

Adenylate cyclase system of bovine adrenal plasma membranes.

The adenylate cyclase system present in a preparation enriched in plasma membranes derived from bovine adrenal cortex was investigated in considerable detail. This system is stimulated by adrenocorticotropic hormone (ACTH), by biologically active analogs of this hormone, and by fluoride ion. The preparation contains sodium-potassium- and magnesium-dependent ATPases that are markedly inhibited by 50 mM sodium fluoride. Incorporation of a pyruvate phosphokinase ATP generating system into the adenylate cyclase assay medium provided constant substrate levels. In the presence of the ATP generating system, the rate of cyclic AMP formation (basal, fluoride, and ACTH-activated) was proportional to enzyme concentration and was linear with time. Proportionality with respect to enzyme concentration as concerned the hormone-activated adenylate cyclase was achieved only when the ratio of hormone to enzyme protein was kept constant. The temperature optimum of the adenylate cyclase, basal or activated, was approximately 30 degrees. Michaelis-Menten kinetics were observed when the ratio of Mg2+ to ATP was approximately 6:1. Both calcium and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid completely inhibited the adenylate cyclase system at concentrations of 5 and 0.5 mM, respectively. GTP was inhibitory at concentrations of 10-2 M but had little effect at lower concentrations. Freezing in liquid nitrogen and storage at -60 degrees exerted little effect on the fluoride-stimulated enzyme but lowered hormone stimulated activity. Preincubation in the presence of ACTH afforded a high degree of stabilization of the enzyme system while preincubation with a biologically inactive analog afforded no protection.

Adenosine Triphosphatases↗
PubMed1975

Lipids of bovine adrenal plasma membranes.

The lipid composition of a membrane fraction from bovine adrenal cortex was determined. This preparation has the capacity to bind adrenocorticotropic hormone and is enriched in adenylate cyclase and 5'-nucleotidase. The adrenal plasma membranes have a significantly higher lipid content (54.8%) than bovine liver plasma membranes and a surprisingly low proportion of this lipid is cholesterol (4.2%). The phospholipids comprise 76.4% of the total lipids and their composition if very similar to that of bovine liver membranes with the exception of sphingomyelin which comprises only 4.5% of the phospholipids in the adrenal preparation.

Adenylyl Cyclases↗
PubMed1974

ACTH antagonists.

Structural modifications within the active site of the ACTH molecule have produced analogs that inhibit the hormone sensitive adenylate cyclase system of bovine adrenal cortical plasma membranes. It is demonstrated that the tryptophan residue of the ACTH molecule is essential for stimulation of the enzyme. Substitution of tryptophan by phenylalanine or by N(alpha)-methyltryptophan as in [Gln(5), Phe(9)]corticotropin(1-20) amide or [N(alpha)-Metrp(9)]corticotropin(1-24) provides ACTH analogs that exhibit high affinity for the ACTH receptor(s) but fail to activate the adenylate cyclase system. It is concluded that affinity for the receptors alone is not sufficient for expression of hormonal activity. The observation that adrenal cortical adenylate cyclase activated by fluoride ion is not inhibited by the antagonists indicates that hormonal and fluoride activation proceed via different mechanisms.

Adenylyl Cyclases↗