Dendrotoxin-binding brain membrane protein displays a K+ channel activity that is stimulated by both cAMP-dependent and endogenous phosphorylations

The purified protein that binds the K+ channel ligands dendrotoxin I and mast cell degranulating peptide can be phosphorylated by cAMP-dependent protein kinase and by an endogenous protein kinase, which may be a specific K+ channel kinase. Phosphorylations take place on the toxin-binding subunit, a polypeptide of 76-80 kDa. Phosphorylation by both kinases leads to activation of the reconstituted dendrotoxin-sensitive K+ channel.     

Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. Evidence from the N-terminal sequence of its larger subunit

High affinity acceptors for alpha-dendrotoxin, a selective probe for certain fast activating voltage-dependent K+ channels, were purified approximately 4,000-fold from synaptic plasma membranes of bovine cerebral cortex. Although the preparation possessed a low content of high affinity sites for beta-bungarotoxin, antagonism of alpha-dendrotoxin binding by the latter required high concentrations; this indicates that more than one acceptor subtype has been purified. After deglycosylation of the acceptor, the sizes of its subunits were determined electrophoretically to be Mr 65,000 and Mr 39,000. Solid phase microsequencing of these isolated subunits showed that the smaller one had a blocked N terminus, but the Mr 65,000 protein gave a sequence of 27 residues. This is virtually identical to the N-terminal sequence deduced from cDNA of RCK 5, a K+ channel protein from rat brain known to be susceptible to alpha-dendrotoxin. This first report on the partial sequence of any K+ channel protein confirms that the extensive information acquired to date on the alpha-dendrotoxin acceptors is pertinent to functional neuronal K+ channels.     

Vascular smooth muscle expresses a truncated Na+, K(+)-ATPase alpha-1 subunit isoform.

By regulating transmembrane Na+ and K+ concentrations and membrane potential, the Na+,K(+)-ATPase plays an important role in regulating cardiac, skeletal, and smooth muscle function. A high degree of amino acid sequence and structural identity characterizes the three Mr 100,000 Na+,K(+)-ATPase alpha subunit isoforms expressed in cardiac and skeletal muscle. Strikingly, vascular smooth muscle utilizes alternative RNA processing of the alpha-1 gene to express a structurally distinct Mr approximately 65,000 isoform, alpha 1-T (truncated). Analysis of both its mRNA and protein structure reveals that alpha-1-T represents a major, evolutionarily conserved, truncated Na+,K(+)-ATPase isoform expressed in vascular smooth muscle. This demonstrates an unexpected complexity in the regulation of vascular smooth muscle Na+,K(+)-ATPase gene expression and suggests that a structurally novel, truncated alpha subunit may play a role in vascular smooth muscle active ion transport.     

Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the alpha subunit of the vertebrate enzyme.

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.     

Organization of the F0 sector of Escherichia coli H+-ATPase: the polar loop region of subunit c extends from the cytoplasmic face of the membrane

The membrane-spanning F0 sector of the Escherichia coli H+-transporting ATP synthase (EC 3.6.1.34) contains multiple copies of subunit c, a 79 amino acid residue protein that is thought to insert in the membrane like a hairpin with two membrane traversing alpha-helices. The center of the protein is much more polar than the putative transmembrane alpha-helices and has been postulated to play a crucial role in coupling H+ translocation through F0 to ATP synthesis in the membrane extrinsic, F1 sector of the complex. However, the direction of insertion of subunit c in the membrane has not been established. We show here that the "polar loop" lies on the F1 binding side of the membrane. A peptide corresponding to Lys34----Ile46 of the polar loop was synthesized. Antisera were generated to the Lys34----Ile46 cognate peptide, and the polyclonal antipeptide IgG was shown to bind to a crude F0 fraction by using enzyme-linked immunosorbent assays. The antipeptide serum did not bind tightly enough to F0 to disrupt function. However, a polyclonal antiserum made to purified, whole subunit c was shown to block the binding of F1 to the F0 exposed in F1-stripped membranes. Incubation of the antisubunit c serum with the peptide reduced the inhibitory effect of the antiserum on the binding of F1 to F0. The reversal of inhibition by the peptide was specific to the antisubunit c serum in that the peptide had no effect on inhibition of F1 binding to F0 by antiserum to subunit a of F0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of the 9-10S estradiol receptor from calf uterus

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.     

Binding of dicyclohexylcarbodiimide to a native F1-ATPase-inhibitor protein complex isolated from bovine heart mitochondria

The effect and the binding of dicyclohexylcarbodiimide (DCCD) to a soluble native F1-ATPase-inhibitor protein complex (F1-IP) isolated from heart mitochondria was studied. About one mol DCCD bound per mol F1-IP complex; this inhibited its ATPase activity by more than 95%, ever under conditions that led to maximal hydrolysis. Bound DCCD localized to beta-subunits of the F1-IP complex. Cross-linking of the DCCD labeled complex with N-(ethoxy-carbonyl)-2-ethoxydihydroquinoline yielded a protein with a Mr 65,000-67,000 that contained IP as evidenced by its reaction with IP antibodies. No alpha-subunits were detected in this cross-linked product. The Mr 65,000-67,000 protein corresponds to beta-subunits cross-linked with IP (Klein et al, Biochemistry 1980; 19, 2919-2925). However, no DCCD was found in the cross-linked beta-subunit-IP product of labeled native F1-IP. Thus the beta-subunit in contact with IP is distinct from the other two beta-subunits of the enzyme.     

Isolation of the native form of chicken gizzard myosin light-chain kinase.

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.     

Functional effects and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase

Subunit b (uncF protein) of the proton-ATPase (F1F0) of Escherichia coli was purified from membranes of strain AN1460 (unc+). Antibody to purified subunit b was raised in rabbits. It reacted with F1-depleted membranes and blocked F1 binding. Bound antibody had no effect on proton transport through F0. F1-Depleted membranes competed with purified subunit b for antibody in an enzyme-linked immunosorbent assay. F1-Depleted membranes which had been pretreated with trypsin or preincubated with saturating amounts of soluble F1 competed poorly with purified subunit b for antibody. The antibody to subunit b was used to further evaluate the trypsin-cleavage data previously reported [D. S. Perlin, D. N. Cox, and A. E. Senior (1983) J. Biol. Chem. 258, 9793-9800]. The results indicated that trypsin proteolysis of F1-depleted membranes resulted in the transient appearance of three fragments of subunit b (Mr = 16,400, 15,700, and 15,500) that remained tightly bound to the membrane. A water-soluble fragment (Mr 14,800), previously thought to be derived from subunit b, was not detected by the antibody. The antibody to subunit b did not cross-react with any subunit of mitochondrial, chloroplast, or other bacterial proton-ATPase, or with the proton-ATPase of clathrin-coated vesicles, plant microsomal membranes, or Neurospora crassa plasma membranes.     

Phosphorylation of brain muscarinic receptor: evidence of receptor regulation

Muscarinic receptor, from porcine synaptic membrane, was purified by affinity chromatography. Molecular weight analysis by SDS-gel electrophoresis revealed one major peptide with an apparent Mr of 68 +/- 2 Kda. The purified receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase resulting in a concomitant loss in specific binding, and this loss was reversed by calcineurin.     

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.     

Isolation and characterization of two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.     

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

Purification of a latent protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.     

Purification of the N,N'-dicyclohexylcarbodiimide-binding proteolipid of a higher plant tonoplast H+-ATPase

The H+-ATPase of Beta vacuolar membrane (tonoplast) comprises at least three functionally distinct subunits of Mr = 67,000, 57,000, and 16,000, respectively (Manolson, M. F., Rea, P. A., and Poole, R. J. (1985) J. Biol. Chem. 260, 12273-12279). The hydrophobic carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the enzyme with pseudo-first order kinetics, and the concentration dependence of the reaction indicates that DCCD interacts with a single site on the enzyme to exert its inhibitory effect. The apparent pseudo-first order rate constant (k0) is reciprocally dependent on membrane protein concentration, which is expected if a large fraction of the DCCD partitions into the lipid phase. k0 has a nominal value of 1000 M-1 min-1 at a protein concentration of 250 micrograms/ml, although when phase partitioning is taken into account, the true, protein concentration-independent value of k0 is calculated to be about an order of magnitude lower. [14C]DCCD primarily labels the Mr = 16,000 polypeptide of native tonoplast vesicles. Binding is venturicidin-insensitive and occurs at a rate similar to the rate of enzyme inactivation, implying that inhibition is a direct result of covalent modification of the Mr = 16,000 polypeptide. Labeling of the containing Mr = 8,000 subunit of mitochondrial F0F1-ATPase is, on the other hand, faster by a factor of 5 and totally abolished by venturicidin. These results confirm that the Mr = 16,000 polypeptide which copurifies with tonoplast H+-ATPase activity is a subunit of the enzyme. Most of the DCCD-reactive Mr = 16,000 subunit is extracted from acetone:ethanol-washed tonoplast vesicles by chloroform:methanol. [14C]DCCD bound to the Mr = 16,000 polypeptide is enriched in the chloroform:methanol extract by 5-fold compared with native tonoplast and the specific activity (nmol of [14C]DCCD/mg of protein) can be increased a further 37-fold by chromatography on DEAE-Sephadex. It is concluded that the Mr = 16,000 subunit of the tonoplast H+-ATPase is a proteolipid.     

Selenium-containing proteins of rat kidney

Rat kidney selenium (Se)-containing proteins were studied by isotopic labeling with [75Se]selenite or [75Se]selenomethionine via three routes: oral, intraperitoneal injection, and incubation of kidney slices with the isotope. The two major Se-containing proteins in kidney were fractionated and partially characterized. 75Se elution profiles from Sephadex G-150 chromatography were similar for each labeling protocol, except for the profile obtained following incubation of slices with [75Se]selenomethionine. Of the two major 75Se-containing proteins, the one eluting at the void volume during Sephadex G-150 fractionation had a subunit of 23,000 Mr. The 75Se-labeled tryptic peptide from this protein and a 75Se-containing tryptic peptide from glutathione peroxidase had the same elution time from an HPLC column. A 75,000 Mr 75Se-containing protein had a 65,000 Mr subunit, and the 75Se-labeled tryptic peptide from this protein eluted from the HPLC column before that of glutathione peroxidase. Glutathione peroxidase is the most abundant kidney selenoprotein. Injection of animals with 75Se is the method of choice for isotopic labeling of rat kidney Se-containing proteins. Appropriate methods were developed that can be used in future studies of kidney Se-containing proteins.     

Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.     

The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport

The (Na+ + K+)-ATPase of cultured chick sensory neurons was studied with the aid of antibodies specific for this enzyme. Immunofluorescent labeling indicated the (Na+ + K+)-ATPase is evenly distributed on the neuronal cell surface; cell bodies, neurites, and growth cones were labeled with comparable intensity. Pulse-chase experiments with [35S]methionine, followed by immunoprecipitation, indicated concurrent synthesis and rapid association of the alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. The alpha subunit is oligosaccharide-free while the beta subunit contains three Asn-linked oligosaccharide chains attached to a core peptide of 32,000 molecular weight. The time required for oligosaccharide processing of the newly synthesized beta subunit to endoglycosidase H-resistance suggests the (Na+ + K+)-ATPase takes 45-60 min to move from the site of polypeptide synthesis to the Golgi apparatus. Significantly less time was required for transport through the Golgi apparatus and insertion in the plasma membrane. From 30% to 55% of the newly synthesized (Na+ + K+)-ATPase did not appear on the cell surface but accumulated intracellularly. When tunicamycin was used to inhibit glycosylation of the beta subunit, there was no effect upon subunit assembly, intracellular transport, or degradation rate (t1/2 = 40 h).     

Differential effect of ethanol on formation of phosphorylated intermediates of the jejunal brush border alkaline phosphatase Mr 90,000 and 65,000 subunits

The influence of ethanol on formation of phosphorylated intermediates of different forms of alkaline phosphates in brush border of adult rat was studied. Ethanol enhanced phosphorylation of the Mr 65,000 subunit of jejunal enzyme but had no effect on the Mr 90,000 subunit. Commercial highly purified alkaline phosphate in the presence of ethanol also displayed a two times higher phosphorylation ability.