Rapid cation flux from Torpedo californica membrane vesicles: comparison of acetylcholine receptor enriched and selectively extracted preparations.

Rapid efflux of ²²Na from within closed vesicles derived from $\text{Torpedo}\text{californica}$ electroplax membranes has been studied as an $\text{in}\text{vitro}$ assay of acetylcholine receptor functionality. The most highly purified membrane preparations contained major polypeptides of M.W. 43 and 90 × 10³ daltons in addition to the four peptides characteristic of the acetylcholine receptor (40, 50, 60, 65 × 10³ daltons). Removal of these extra peptides by base extraction did not significantly alter the characteristics of carbamylcholine induced ²²Na efflux: the agonist dose response curve was similar, preequilibration with agonist caused desensitization, the irreversible antagonist α-Bungarotoxin blocked the efflux and the reversible blockade by the neurotoxin perhydrohistrionicotoxin was also retained. The dose response curve for perhydrohistrionicotoxin corresponded closely to its known binding characteristics for base extracted membranes.     

Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. β-d-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside or Triton X-100-solubilized extracts exhibits identical amino acid compositions, α-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530 000 $\text{M}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc.Natl. Acad. Sci. U.S.A. 77, 1796–1799).Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization, purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000-6000 pmol per mg protein of alpha-[125I]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius 125 +/- 10 A and on sucrose gradient centrifugation one major peak was observed of 20-22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Detection of nicotinic cholinergic transmission in malapteruruselectricus electrop lax

Biochemical and electrophysiological studies were conducted on the electric organ of the electric fish of the Nile, $\text{Malapterurus}$$\text{electricus}$, in order to determine if transmission was chemically mediated. There was no binding of [³H] acetylcholine, [³H] quinuclidinyl benzilate or [³H]-perhydrohistrionicotoxin; but low acetylcholinesterase activity was observed, as was binding of [¹²⁵I] α-bungarotoxin. The latter binding was detectable at 0.85 ± 0.07 pmol/g tissue, and was totally inhibited by 1 μM α-bungarotoxin or 100 μM d-tubocurarine. A tetrodotoxin-sensitive action potential was measured which was Na⁺- dependent. Depolarization (30–40 mV) was caused by carbamylcholine, and this was blocked by d-tubocurarine or α-bungarotoxin. The data suggest that this electric organ which may be a rich source for electrically excitable channels, is innervated by nicotonic cholinergic motoneurons, but the concentrations of acetylcholine receptors and acetylcholinesterase are very low.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

Carbamylcholine-induced rapid cation efflux from reconstituted membrane vesicles containing purified acetylcholine receptor.

Membrane preparations containing essentially only the four polypeptides considered to constitute the acetylcholine receptor are purified from $\text{Torpedo}\text{californica}$ electroplax. Treatment of these membranes with 2% ($\text{w}\text{v}$ aqueous sodium cholate followed by removal of all insoluble matter results in a solubilized purified receptor preparation that can be reassociated with phospholipids during dialysis to remove the detergent. Such reconstituted receptor is shown to retain the capability of translocating ²²Na⁺ across the membrane in response to carbamylcholine binding in a highly reproducible manner. The dose response for this effect is similar to that observed for the original electroplax membrane preparation and the carbamylcholine induced signal is completely blocked by α-bungarotoxin.     

A membrane glycoprotein which binds antilymphocyte globulin and concanavalin A is implicated in stimulation of murine T lymphocytes.

Murine thymus cells respond $\text{in vitro}$ to horse anti-lymphocyte globulin (ALG) by incorporation of ¹²⁵IdU, while spleen cells from nu/nu mice are unresponsive. However, absorption of ALG with both nu/nu spleen cells and thymocytes reduces its mitogenic activity. All of the major iodinated surface proteins recognized by ALG bind to insolubilized Concanavalin A; non-mitogenic levels of ALG block Con A stimulation of thymocytes and vice versa. A membrane glycoprotein with an apparent molecular weight on SDS-polyacrylamide gels of 150–200, 000 is proposed as the receptor of ALG-mediated activation of T cells and as a functional Con A receptor.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormones and labelled antibody binding techniques

The parathyrin receptor in renal cortex has been investigated by studying the binding of ¹²⁵I-labelled parathyrin, or of unlabelled parathyrin detected with ¹²⁵I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 °C but this phenomenon was not detectable at 37 °C. Specific displacement of lactoperoxidase labelled ¹²⁵I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was $\text{2.3 · 10}{}^8\text{M}{}^{\mathrm{?1}}$ and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0–7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1–34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glueagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10^?8 M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10^?8 M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to $\text{3}\text{4}$ of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about $\text{3}\text{4}$ of that of untreated cytosol.     

Reiteration frequency of procollagen genes in the guinea pig genome: Collagen genes are not amplified during granuloma fibroblasts differentiation

Procollagen mRNA was purified from collagen synthesizing polysomes obtained from an experimental guinea pig granuloma, and iodinated in vitro. The procollagen ¹²⁵I-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot $\text{1}\text{2}$ 800–900 mol · s · l^?1 for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11–13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplification in tissues highly specialized in the synthesis of collagen such as granuloma.     

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of ¹²⁵I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of ¹²⁵I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K_d of 1·10^?8 M. Nicotine and acetylcholine iodide at concentrations of 10^?5 M inhibit ¹²⁵I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10^?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10^?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.     

Isolation of a 110 000 molecular weight protein in the purification of the nuclear chick intestinal 1,25-dihydroxyvitamin D-3 receptor

A single protein band of molecular weight 110 000 has been obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D-3) receptor from crude nuclear extracts of chick intestinal mucosa, prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride and ?-aminocaproic acid. The nuclear extract was subjected to a six-step purification scheme, involving polymin P and ammonium sulfate fractionation, DNA-cellulose affinity chromatography, Sephacryl S-200 gel filtration, blue dextran-Sepharose and a final DNA-cellulose chromatographic step. The receptor was obtained in about 1% yield and was purified approx. 3700-fold from the nuclear extract, as assessed by specific activity. Single peaks were observed with ³H-1,25-(OH)₂D-3-labeled crude nuclear extracts on Sephacryl S-200 gel filtration ($\text{Strokes′ radius}\text{= 35.5}\text{A}\text{?}$) and sucrose density gradient centrifugation (3.5 S). Although the identity of the M_r 110 000 protein will remain inconclusive until methods for further characterization are available, it may represent evidence for a higher molecular weight form of the 1,25-(OH)₂D-3 receptor than that observed previously.     

Development of the neuromuscular junction in the chick embryo: The number,distribution, and stability of acetylcholine receptors

The number, distribution, and stability of skeletal muscle acetylcholine receptors during development of the neuromuscular junction in the chick embryo were studied. The distribution and turnover of receptors labeled with ¹²⁵I-labeled α-bungarotoxin were determined by quantitative autoradiography on individual teased muscle fibers. Each posterior latissimus dorsi muscle fiber, which in the adult is singly innervated, has a high density of acetylcholine receptors at a single spot from embryonic Day 10 through hatching. The spots stain more intensely than elsewhere for acetylcholinesterase and are assumed to be end plates. The receptors at these spots are presumed to be junctional receptors. The junctional receptor density remains constant at 10⁴/μm² from embryonic Day 14 through adult life, although the area of the junction increases 40-fold. In contrast, the extrajunctional receptor density drops precipitously from 250/μm² on Day 14 to only 10/μm² on Day 19. This decrease in extrajunctional receptor density can be prevented by chronic paralysis with curare. The rate of autoradiographic grain loss from junctional and extrajunctional regions after a pulse injection of ¹²⁵I-labeled α-bungarotoxin indicates that both classes of embryonic receptors turn over at the same rate ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 30}\text{hr}$).     

Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture

Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from $\text{> 200 000}\text{to}\text{12 500}$. Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95 000 and 78 000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[¹²⁵I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Fractionation by velocity sedimentation of Torpedo nicotinic post-synaptic membranes

Velocity sedimentation on sucrose gradients containing Torpedo physiological saline has been utilized to fractionate Torpedo (Torpedo californica and T. nobiliana) post-synaptic membranes isolated initially on the basis of their density by equilibrium centrifugation. Membranes are separated into two populations: (1) those retained within the gradient (referred to as gradient pool); and (2) membranes sedimenting rapidly through the gradient (referred to as f 22, fraction 22 of the gradient). Comparison of their polypeptide compositions by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicates that the gradient pool consists of highly purified nicotinic post-synaptic membranes containing the peptides of the acetylcholine receptor and a peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000, while f 22 contains the contaminating membranes present in the initial suspension as well as a small fraction of the nicotinic post-synaptic membranes. On the basis of the kinetics of efflux of ²²Na⁺ from the membrane fractions, it is concluded that the gradient pool contains most of the sealed vesicles with functional nicotinic receptors. The internal volume (μl/mg protein) of those membranes exceeds that of f 22 by a factor of 4, and greater than 85% of that internal volume is equilibrated by the nicotinic agonist carbamylcholine, while for f 22 only 40% is equilibrated. Thin-section electron microscopy has been used to estimate the distribution of vesicle sizes. The observed distribution for the gradient pool indicates that these vesicles are a size homogeneous population of diameter 0.3 μm, while f 22 contains a number of smaller and larger vesicles. Torpedo post-synaptic membranes have been treated with alkali to remove the non-receptor peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000. After alkaline extraction, velocity sedimentation permits the isolation of a population of size-homogeneous and well-sealed vesicles containing only the peptides of the nicotinic receptor. It is concluded that upon homogenization, the innervated surface of the Torpedo electroplax tends to form vesicles of uniform size (0.3 μm) which can be readily isolated by velocity sedimentation and that the peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000 is not required for the maintenance of bilayer structure.     

Molar extinction coefficients and other properties of an improved reaction center preparation from Rhodopseudomonas viridis

Reaction centers have been purified from chromatophores of Rhodopseudomonas viridis by treatment with lauryl dimethyl amine oxide followed by hydroxyapatite chromatography and precipitation with ammonium sulfate. The absorption spectrum at low temperature shows bands at 531 and 543 nm, assigned to two molecules of bacteriopheophytin b. The 600 nm band of bacteriochlorophyll b is resolved at low temperature into components at 601 and 606.5 nm. At room temperature the light-induced difference spectrum shows a negative band centered at 615 nm, where the absorption spectrum shows only a weak shoulder adjacent to the 600 nm band. The fluorescence spectrum shows a band at 1000 nm and no fluorescence corresponding to the 830 nm absorption band. Two molecules of cytochrome 558 and three of cytochrome 552 accompany each reaction center. The differential extinction coefficient (reduced minus oxidized) of cytochrome 558 at 558 nm was estimated as 20 ± 2 mM^?1 · cm^?1 through a coupled reaction with equine cytochrome c. The extinction coefficient of reaction centers at 960 nm was determined to be 123 ± 25 mM^?1 · cm^?1 by measuring the light-induced bleaching of P-960 and the coupled oxidation of cytochrome 558. The corresponding extinction coefficient at 830 nm is 300 ± 65 mM^?1 · cm^?1. The absorbance ratio $\text{a}{}_{\mathrm{<mtext>280</mtext><mtext>nm</mtext>}}\text{a}{}_{\mathrm{<mtext>830</mtext><mtext>nm</mtext>}}$ in our preparations was 2.1, and there was 190 kg protein per mol of reaction centers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major components of apparent molecular weights 31 000, 37 000 and 41 000.     

Author index

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as $\text{p-}\text{azido}\text{-m-[}{}^{\mathrm{<mtext>125</mtext><mtext>I</mtext><mtext>]</mtext><mtext>iodobenzylcarazolol</mtext>}}\text{. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su?}$ variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.