Isoelectric focusing of brain adenylate cyclase

Mouse brain adenylate cyclase has been solubilized with Lubrol PX and separated by isoelectric focusing on polyacrylamide gels. The enzyme activity has been measured with a sensitive assay isolating cyclic AMP from Dowex and alumina columns. The technique allows a one-step analysis of this membrane enzyme from a heterogeneous sample within 6 hr.     

Preparative isoelectric focusing in agarose

The use of agarose gels as supporting media for flat-bed preparative isoelectric focusing was applied to the fractionation of serum proteins in the pH range 3.5–6, and red cell hemolysates in the pH range 3–8. The agarose gels are easy to prepare, give linear pH gradients, and do not appear to produce molecular sieving effects. Up to 1 g serum proteins can be loaded on the gels, with recoveries between 68 and 82%. Nucleoside phosphorylase from red cell lysates was recovered with 76% yield, indicating that no appreciable denaturation of this enzyme had occurred. Preparative isoelectric focusing in agarose gels provides a useful alternative to existing techniques of preparative isoelectric focusing in sucrose gradients or granulated gels.     

Neurotoxic esterase: gel filtration and isoelectric focusing of carboxylesterases solubilized from hen brain

Carboxylesterase activity (EC 3.1.1.1) of hen brain including neurotoxic esterases NTEA and NTEB is solubilized from lyophilized lipid-extracted brain material by the use of n-octylglucoside. The solubilized enzymes are subjected to free isoelectric focusing, six carboxyl - esterase activity peaks are obtained. By gel filtration on Sephacryl S-300 neurotoxic esterases are separated from carboxylesterase isoenzymes V and X. The molecular weight of the neurotoxic esterases is estimated to be 1.8 X 10(6).     

Activation by cholera toxin of adenylate cyclase solubilized from rat liver.

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.     

Preparative isoelectric focusing with Pevikon as supporting medium

A preparative isoelectric focusing method is described in detail that uses Pevikon instead of Sephadex as a supporting medium. Separation is demonstrated in a human serum protein preparation that contained α₁-antitrypsin, transferrin and α₂-macroglobulin. Pevikon has some advantages over Sephadex in this type of isoelectric focusing.     

Bovine adrenocortical adenylate cyclase: Some properties of the solubilized,fluoride-activated enzyme

The activity of bovine adrenocortical plasma membrane adenylate cyclase can be maintained at 4°C in the presence of NaF. The half-life of the fluoride-stabilized enzyme is approximately 7 days. Maximal activation by fluoride requires approximately 20 min at 0°C and the level of activity attained is dependent on fluoride concentration. The enzyme from freshly harvested membranes can also be stimulated by ACTH_{1 – 24} and Gpp(NH)p and the stimulatory effects of these two activators are additive. Prolonged exposure to either NaF or Gpp(NH)p precludes hormone activation. Optimal concentration for Gpp(NH)p activation is 10^?4–10^?5M. Treatment of the enzyme with a Tris-HCl buffer containing Lubrol-PX (1%), NaF, dithiothreitol, and MgSO₄ followed by sonication affords a preparation that does not sediment at 100 000 g in 1 hr. This material has a low specific activity; however, removal of the detergent on DEAE cellulose restores specific activity to its original level. A significant improvement in specific activity is observed following dialysis or ultrafiltration of the detergent-free 100 000-g supernatant. At this stage the enzyme can be lyophilized and stored at ?70°C without loss of activity. The enzyme in the detergent-free 100 000-g supernatant behaves as a single peak that is included in Sepharose 6B. Comparison of the elution profile of the enzyme with profiles produced by a standard set of proteins suggests a molecular weight of 1 × 10⁶. Hydrophobic chromatography of the detergent-free 100 000-g supernatant on n-hexyl Sepharose 4B results in a fivefold enhancement of specific activity.     

Activation of solubilized liver membrane adenylate cyclase by nucleotides

Liver plasma membranes isolated from hypophysectomized rats were treated with 0.1 M Lubrol-PX, a nonionic detergent, and centrifuged at 165,000 × g for 1 hour. Adenylate cyclase activity remaining in the supernate had a specific activity that was at least equal to that of the particulate enzyme. The activity of the solubilized, non-sedimentable adenylate cyclase, as well as the membrane bound enzyme, was increased by GTP, ITP, and GMP-PCP at 10^?4 M. The activity of the solubilized, non-sedimentable enzyme increased linearly with GTP from 10^?6 to 10^?4 M but there was no further increase in the activity of the solubilized enzyme with 10^?3 M GTP. In contrast, the particulate liver membrane enzyme activity increased exponentially with GTP from 10^?6 to 10^?4 M and was further increased by 10^?3 M GTP. These data indicate that GTP, ITP or GMP-PCP have direct effects on solubilized adenylate cyclase. This effect is in addition to a role of nucleotides in modifying membrane structure (16).     

Galanin inhibits adenylate cyclase of rat brain membranes.

The ubiquitous neuropeptide, galanin, strongly inhibits adenylate cyclase in rat brain membranes. While basal enzyme activity was not altered, galanin from 10(-11) M to 5 x 10(-7) M decreased forskolin- and VIP-stimulated adenylate cyclase with a half-maximal effect being elicited by 0.7 nM neuropeptide and a maximal 80% inhibition of the enzyme activity. The galanin fragments (2-29) and (1-15) dose-dependently inhibited the forskolin-stimulated adenylate cyclase, while the fragments (3-29) and (10-29) were found inactive. These results indicate that the regulatory action of galanin in the central nervous system involves the coupling of galanin receptors to the inhibition of the adenylate cyclase system.     

Effect of undernutrition on GMP-PNP binding and adenylate cyclase activity from rat brain

1. Undernutrition is an insult that affects brain development and functioning. Considering that signaling through metabotropic receptors/G proteins is critical for normal synaptic transmission and contributes to CNS development and synaptic plasticity, the present study investigated the effects of pre- and postnatal protein deprivation (diet: 8% protein; normonourished group: 25% protein) on brain signal transduction by G proteins.2. Undernutrition decreased the [³H] GMP-PNP binding to G proteins and AC activity, in neural plasma synaptic membranes of 21- and 75-day-old rats. This effect was less pronounced or even absent in old rats.3. Ontogenetically, the dietary treatment effect might be interpreted as a retarded development associated with protein malnutrition.     

Phosphatidylserine vesicles increase rat brain synaptosomal adenylate cyclase activity

Phosphatidylserine vesicles incubated in hypotonic conditions with rat brain synaptosomes increased basal adenylate cyclase activity but did not modify the response of the enzyme to norepinephrine. Moreover, phosphatidylserine antagonized the stimulation of adenylate cyclase activity by NaF. We suggest that in present experimental conditions the effect of phosphatidylserine vesicles is at the level of the GS regulatory protein of adenylate cyclase.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).     

Properties of adenylate cyclase solubilized from rat adrenal membranes: effects of ACTH and other stimulators on solubilization.

We have solubilized adenylate cyclase in a relatively stable form from rat adrenal membranes. The solubilized enzyme elutes on a column of Sepharose 4BR as a distinct peak with a higher molecular weight than the soluble fractions which bind 125I-ACTH. Both the soluble and membrane bound enzymes are activated by NaF and Gpp(NH)p, and both have similar affinities for MgATP. While the membrane bound enzyme is activated similarly by either Mg2+ or Mn2+, the soluble enzyme is more fully activated by Mn2+. Pretreatment of adrenal membranes with NaF or Gpp(NH)p before the addition of detergent enhances recovery of soluble enzyme activity, while recovery of activity in the unsolubilized membrane pellet is unchanged. In contrast, addition of ACTH prevents solubilization of the enzyme and greatly increases its recovery in the pellet. This observation is consistent with the theory that action of the hormone on a receptor subunit leads to an association between the receptor and a catalytic subunit. Such an association might make it more difficult to remove the enzyme from the surrounding lipid matrix of the membrane.     

Isolation of specific antigens from Angiostrongylus cantonensis. 1. Preparative flatbed isoelectric focusing

Electrophoresis on SDS gel and analytical isoelectric focusing showed that a crude extract of Angiostrongylus cantonensis consisted of at least 40 protein components with molecular weights ranging from 13 000-70 000 and isoelectric points of pI values ranging from 3.7-10.0. Crossed-immunoelectrophoresis with a hyperimmune antiserum to A. cantonensis showed at least 40 different antigenic components in the crude worm extract which were cross-reactive with those of Ascaris suum, Metastrongylus apri and Toxocara canis. Using preparative isoelectric focusing, the somatic worm preparation was divided into 13 equal fractions, of which 3, 4 and 5, with pI values of 3.7, 4.0 and 4.45 respectively, were later shown by immunoelectrophoretic techniques and enzyme-linked immunosorbent assay to contain antigens specific to A. cantonensis.     

Preparative isoelectric focusing of immunologically reactive components of Aspergillus fumigatus mycelium

A water-soluble mycelial extract of Aspergillus fumigatus has been fractionated by preparative isoelectric focusing using carrier ampholytes in a layer of granulated gel. The separated components were located by staining paper prints from the gel. Within a narrow pH range of 2.5 units, multiple protein bands were visualized with Coomassie Brilliant Blue G. Periodate-Schiff-positive material was generally associated with the major protein zones. When these fractions were eluted the total recovery, calculated on the basis of protein and carbohydrate analyses of the isolated fractions, varied between 20 and 60% of the applied material. Low recoveries were associated with low recoveries of protein; recoveries of carbohydrate were higher and less variable. The immunological activity and specificity of the eluted fractions were assessed in an enzyme-linked immunosorbent assay for the detection of IgG antibodies to A. fumigatus.     

Activation of adenylate cyclase by forskolin in rat brain and testis

Detergent-dispersed adenylate cyclase from rat cerebrum was detected in two components, one sensitive to Ca2+ and calmodulin and another sensitive to fluoride or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The enzyme activity of both components was markedly augmented by forskolin assayed in the presence or absence of other enzyme activators (e.g., NaF, Gpp(NH)p, calmodulin). The catalytic subunit fraction in which G/F protein was totally lacking was also activated by forskolin. During 1-35 days of postnatal development, the basal adenylate cyclase activities in either cerebrum and cerebellum particulate preparations progressively increased. While the fluoride sensitivity of the cerebrum and cerebellum enzyme increased during postnatal development, the responsiveness to forskolin remained unaltered. There was no enhancement of soluble adenylate cyclase (from rat testis) by forskolin under the assay conditions in which there was a marked stimulatory action on the particulate enzyme. The results seen with the solubilized enzyme, with either Lubrol PX or cholate, indicate that the effects of forskolin on the cyclase do not require either G/F protein or calmodulin and the results of our study of brain enzymes support this view. Data on soluble testis cyclase (a poor or absent response to forskolin by this enzyme) imply that it lacks a protein (other than the catalytic unit) which could confer greater stimulation. The present results do not rule out an alternative explanation that forskolin stimulates adenylate cyclase by a direct interaction with the catalytic subunit, if the catalytic proteins do differ widely in various species of cells and their response to this diterpene.