Postsynaptic membranes in the electric tissue of Narcine: II. A freeze-fracture study of nicotinic receptor molecules

The ventral, postsynaptic membranes of the electroplaques of Narcine were found to containe intramembranous particles similar in location, packing density (about 5700/micron 2), transmembrane position and end appearance to nicotinic acetylcholine receptor-channel molecules. In fixed tissue the particles were limited to the cytoplasmic lamina, while in unfixed tissue an equivalent number were found symmetrically in both laminae. Four populations of particle diameters were observed in each unfixed lamina, even though other morphological evidence indicates the presence of large number of molecules of uniform structure, and biochemical studies of isolated postsynaptic membranes indicate that at least 70% of the membrane protein is receptor-channel protein. Intramembranous particles in dorsal, non-innervated electroplaque membranes, presumably representing Na+, K+-associated ATPase and other channel proteins, were found to have similar characteristics to particles in ventral membranes. Receptor-channel molecules cannot, therefore, be distinguished from other intrinsic membrane proteins by freeze-replication alone.     

The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis.

Serum from hamsters bearing group C adenovirus-induced tumors can be divided into two classes: first, a broad spectrum serum that contains antibodies to several early adenovirus proteins, immunoprecipitated from virus-infected cell extracts, with molecular weights of 72,000, 58,000, 44,000 and 17,000 daltons; and second, a narrow spectrum serum that contains antibodies to the 58,000 dalton protein from virus-infected cell extracts. Both types of sera have been used to immunoprecipitate specifically the 58,000 dalton protein from a type 2 adenovirus-transformed hamster cell line and a type 2 adenovirus-SV40 nondefective hybrid (Ad2⁺ND-1) transformed hamster cell line. In addition, the broad spectrum serum immunoprecipitates or co-precipitates a late adenovirus protein of 120,000 daltons from virus-infected, but not virus-transformed cells.Peptide maps of the 120,000 dalton antigen and the virus hexon structural protein (120,000 daltons) demonstrate that these proteins are closely related. The 72,000 dalton antigen has been shown to be the adenovirus single-strand-specific DNA binding protein. Peptide maps of this 72,000 dalton antigen demonstrate that it contains all the peptides found in the 44,000 dalton antigen. The 72,000 dalton antigen contains two additional peptide fragments not detected in the 44,000 dalton protein, indicating that this 44,000 dalton antigen is a proteolytic breakdown product of the 72,000 dalton protein. The 58,000 dalton adenovirus tumor antigen has a peptide map which is completely distinct from the 120,000, 72,000 and 44,000 dalton proteins. These data demonstrate that the 58,000 dalton antigen is chemically distinct from the 72,000–44,000 dalton early adenovirus proteins.     

Identification of the primary translation product of atrial natriuretic peptide receptor mRNA in a cell-free system using anti-receptor antiserum

The primary translation product of mRNA encoding atrial natriuretic peptide (ANP) receptor has been shown to have an Mr of 58,000. Poly(A)+ RNA was isolated from the bovine kidney and lung and translated in a rabbit reticulocyte lysate system containing [35S]methionine. Immunoprecipitation of the labeled translation products, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, identified a 58-kDa protein as the primary translation product which is the unglycosylated precursor to be processed to the glycosylated mature 70-kDa form found in the plasma membranes. The result lends strong support to our previous proposal that mature ANP receptor is composed of two disulfide-linked 70-kDa subunits, eliminating the possibility that the two 70-kDa subunits arise from a larger 140-kDa precursor by proteolytic cleavage.     

The Mr 93,000 polypeptide of the postsynaptic glycine receptor complex is a peripheral membrane protein

The glycine receptor of mammalian spinal cord is an oligomeric membrane protein that, after affinity purification on aminostrychnine-agarose or immobilized antibody, contains three polypeptides of Mr 48,000, 58,000, and 93,000. Here, the association and the properties of the polypeptides of the rat glycine receptor were investigated. Upon phase partitioning in the nonionic detergent Triton X-114, the three receptor polypeptides behaved as a hydrophilic protein complex exhibiting phospholipid binding. Sucrose gradient centrifugation or gel filtration in the presence of dithiothreitol and Triton X-100 separated the Mr 93,000 polypeptide from the Mr 48,000 and 58,000 polypeptides, which harbor the antagonist binding site of the glycine receptor. Alkaline or dimethylmaleic acid anhydride treatment of crude synaptic membrane fractions resulted in extraction of the Mr 93,000 polypeptide. Lectin binding was observed for the Mr 48,000 and 58,000 glycine receptor subunits but not the Mr 93,000 polypeptide. These results indicate that the Mr 93,000 polypeptide is a peripheral membrane protein that is located at the cytoplasmic face of the postsynaptic glycine receptor complex.     

Postsynaptic membranes in the electric tissue of Narcine: III. Isolation and characterization

Postsynaptic membranes in homogenates of the electric tissue of Narcine were identified by labelling nicotinic acetylcholine receptors in the membranes with radioactive alpha-bungarotoxin. Various media and centrifugation conditions were examined in an attempt to obtain highly purified postsynaptic membranes. The main criterion for purification was approach towards the specific activity of the pure receptor protein, 9–10 nmol toxin-sites/mg protein. Isolation of tissue microsomes with Tris buffer, EDTA and the protease inhibitor phenylmethylsulfonylfluoride (PMSF), conditions which preserve the receptor molecules optimally, yielded about 50 % of the tissue toxin-sites, 5 % of the protein, 4 % of the ATPase and less than 2 % of the acetylcholinesterase (AChE). Further separation of vesiculated membranes in continuous density gradients of sucrose showed that the major contaminants of postsynaptic membrane vesicles were damaged mitochondria and tubular vesicles of dorsal electroplaque membranes rich in ATPase. Mitochondria were effectively removed from homogenates by ‘differential’ centrifugation, and ATPase-rich vesicles could be largely removed by causing their agglutination with calcium ions, or by controlled proteolysis in the absence of PMSF. Partially purified postsynaptic membranes were obtained having about 7 nmol toxin-sites/mg membrane protein. Further purification appears possible by affinity techniques.     

Postsynaptic membranes in the electric tissue of Narcine: I. Organization and innervation of electric cells. Fine structure of nicotinic receptor-channel molecules revealed by transmission microscopy

The ventral, postsynaptic membranes of the electroplaques of Narcine were examined for structures corresponding in location, numbers, position and form with nicotinic acetylcholine receptor-channel molecules. Electron micrographs of conventionally fixed material showed that the thickened outer lamina of these membranes contained about 6100 stained structures/μm² ; and calculations from this packing density and the estimated area of postsynaptic membranes/g tissue yielded a total number of structures within a factor of two of the number of receptor molecules/g determined from the binding of alpha-bungarotoxin. Examination of tissue prepared with a tannic acid/glutaraldehyde fixative revealed that the structures were uniform in character, cylindrical in shape, transmembrane in position, and that each contained a central core. Their diameter matched that of isolated receptor molecules, and their size can account for most of the receptor protein. We conclude that these receptor molecules are largely intramembranous, cylindrical and exhibit transmembrane pores.     

Deglycosylation and proteolysis of photolabeled D2 dopamine receptors of the porcine anterior pituitary

Dopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr approximately equal to 150,000 and 118,000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and alpha-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mr approximately equal to 44,000, similar to that of deglycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mr = 44,000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.     

Interaction of the 43K protein with components of Torpedo postsynaptic membranes

Interactions of the major Mr 43 000 peripheral membrane protein (43K protein) with components of Torpedo postsynaptic membranes have been examined. Treatment of membranes with copper o-phenanthroline promotes the polymerization of 43K protein to dimers and higher oligomers. These high molecular weight forms of 43K protein can be converted to monomers by reduction with dithiothreitol and do not contain any of the other major proteins found in these membranes, including the subunits of the acetylcholine receptor, as shown by immunoblotting with monoclonal antibodies. To study directly its interactions with the membrane, the 43K protein was radioiodinated and purified by immunoaffinity chromatography. Purified 43K protein binds tightly to pure liposomes of various compositions in a manner that is not inhibited by KCl concentrations up to 0.75 M. The binding can be reversed by adjusting the pH of the reaction to 11, the same treatment that removes 43K protein from postsynaptic membranes. Unlabeled 43K protein solubilized from Torpedo membranes with cholate can be reconstituted with exogenously added lipids in the absence of the receptor. The results suggest that 43K protein molecules are amphipathic and that they may interact with each other and with the lipid bilayer. These interactions cannot explain the coextensive distribution of 43K proteins with acetylcholine receptors in situ. However, they could account for the association of the 43K protein with the postsynaptic membrane and may contribute to the maintenance of the structure of the cytoplasmic specialization of which this protein is a major component.     

Protein phosphorylation of nicotinic acetylcholine receptors

The nicotinic acetylcholine receptor (nAcChR) is a ligand-gated ion channel found in the postsynaptic membranes of electric organs, at the neuromuscular junction, and at nicotinic cholinergic synapses of the mammalian central and peripheral nervous system. The nAcChR from Torpedo electric organ and mammalian muscle is the most well-characterized neurotransmitter receptor in biology. It has been shown to be comprised of five homologous (two identicle) protein subunits (alpha 2 beta gamma delta) that form both the ion channel and the neurotransmitter receptor. The nAcChR has been purified and reconstituted into lipid vesicles with retention of ion channel function and the primary structure of all four protein subunits has been determined. Protein phosphorylation is a major posttranslational modification known to regulate protein function. The Torpedo nAcChR was first shown to be regulated by phosphorylation by the discovery that postsynaptic membranes contain protein kinases that phosphorylate the nAcChR. Phosphorylation of the nAcChR has since been shown to be regulated by the cAMP-dependent protein kinase, protein kinase C, and a tyrosine-specific protein kinase. Phosphorylation of the nAcChR by cAMP-dependent protein kinase has been shown to increase the rate of nAcChR desensitization, the process by which the nAcChR becomes inactivated in the continued presence of agonist. In cultured muscle cells, phosphorylation of the nAcChR has been shown to be regulated by cAMP-dependent protein kinase, a Ca2+-sensitive protein kinase, and a tyrosine-specific protein kinase. Stimulation of the cAMP-dependent protein kinase in muscle also increases the rate of nAcChR desensitization and correlates well with the increase in nAcChR phosphorylation. The AcChR represents a model system for how receptors and ion channels are regulated by second messengers and protein phosphorylation.     

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.     

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.     

An analysis of the structure of the antigen receptor on a pigeon cytochrome c-specific T cell hybrid

The monoclonal antibody A2B4-2 has been shown to bind to the antigen receptor on the cloned pigeon cytochrome c-specific T cell hybrid, 2B4. Initial immunoprecipitation and SDS-PAGE analysis with this clonotypic antibody demonstrated that the antigen receptor on this cell had a m.w. of 85,000 to 90,000. Under reducing conditions, the receptor protein appeared as two bands of 45,000 to 50,000 and 40,000 to 44,000 on an SDS-PAGE gel. In this paper the antigen receptor on this T cell hybrid is further characterized. The molecule is shown to be a heterodimer that exists in two different forms on the cell surface. Receptor molecules with an apparent m.w. of 84,000 and 86,000 were isolated by immunoprecipitation and separation on polyacrylamide gradient gels. After reduction, the individual alpha- and beta-chains were separated by isoelectric focusing. In both forms of the receptor, the acidic alpha-chain had an apparent m.w. of 42,000 to 44,000. This alpha-chain associated with one of two forms of beta-chain. One beta-chain had a m.w. of 42,000 to 44,000, with a pI range of 7.5 to 7.9, and the alternate form of the beta-chain, beta', had a m.w. of 46,000 to 48,000 and a more acidic pI range of 6.5 to 7.5. The results of this investigation indicate that under reducing conditions on SDS-PAGE gels, the original upper 45,000 to 50,000 m.w. band represented beta'-chains alone, whereas the lower 40,000 to 44,000 m.w. band represented a mixture of alpha- and beta-chains. Additional data are presented to indicate that this heterodimeric protein has intrachain as well as interchain disulfide bonds. This conclusion was reached from the characteristic pattern of protein migration on SDS-PAGE gels in the presence of a reducing agent concentration gradient. Thus, both chains of the antigen receptor must have intrachain disulfide bonds and may have similar domain structures.     

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.     

Characterization of the guinea pig adipocyte thyrotropin receptor

125I-TSH binding to porcine thyroid and guinea pig fat resulted in curvilinear Scatchard plots with similar dissociation constants for the high and low affinity binding components. Antibodies from the sera of patients with Graves' disease inhibited binding to the high and low affinity binding components of both tissues. Covalent cross-linking of 125I-TSH to membranes from each tissue resulted in the specific labeling of two protein bands. The guinea pig fat receptor subunits have Mr values of 52,000 and 38,000, whereas the porcine thyroid receptor subunits have values of 46,000 & 35,000. The labeling of the receptor subunits was inhibited by preincubation with Graves' autoantibodies. Despite possessing a different subunit composition, the receptors from these tissues exhibit similar affinity for TSH and share similar antigenic determinants for Graves' autoantibodies.     

Morphology and molecular composition of isolated postsynaptic junctional structures.

The solubilization of isolated brain synaptosomal plasma membranes by various detergents was studied and in each case found to depend upon detergent concentration. By using conditions sufficient to extract maximally protein and phospholipid from the membranes, postsynaptic junctional particles were isolated with each of four detergents and their ultrastructural appearances and protein contents compared. Two basic structural forms were identified. One, isolated with Triton X-100, consists of a planar array of dense-staining particles ca. 20 nm in diameter. It resembles the postsynaptic density seen in undigested synaptosomal plasma membranes. The other, isolated with sodium deoxycholate, contains less protein. It has the same overall shape and dimensions as the postsynaptic density, but consists of a branching network of short 5 nm fibres (the postsynaptic junctional lattice) making up an array of contiguous polygons, each ca. 20 nm across. The interior of these polygonal elements seems to be hydrophobic since it cannot be penetrated by metallic salts used for negative staining. It is suggested that the dense-staining 20 nm subunits observed at the postsynaptic junctional site may be composed of hydrophobic proteins inserted into the hollow cores of the lattice polygons. Electrophoretic analysis of the proteins present in the various postsynaptic junctional preparations identified two major common components with molecular masses of 275000 and 47500. The latter is tentatively identified as actin. Components comigrating respectively with alpha- and beta-tubulin are present, and the relation of the lattice structure to subjacent microtubules is discussed.     

Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.     

Comparison of the properties of the protein phosphatases from avian and mammalian smooth muscles: purification and characterization of rabbit uterine smooth muscle phosphatases

Three protein phosphatases were purified to near homogeneity from rabbit uterine muscle. These enzymes are termed rabbit uterine smooth muscle phosphatase (RU SMP)-I, -II, and -IV. RU SMP-I is composed of three subunits (Mr 60,000, 55,000, and 38,000) which comigrated with the subunits of turkey gizzard smooth muscle phosphatase (TG SMP)-I. Ethanol treatment of RU SMP-I dissociated the subunits and led to the purification of its catalytic subunit (Mr 38,000), RU SMP-Ic. Structural homology between the turkey gizzard and rabbit uterine SMP-I is indicated by the cross-reactivity of RU SMP-I with the polyclonal antibodies against TG SMP-I and -Ic. Like TG SMP-II, RU SMP-II is inactive in the absence of divalent cations and can be activated by Mg2+ and Mn2+. However, their electrophoretic profiles on sodium dodecyl sulfate-polyacrylamide gel are different. RU SMP-II shows two bands (Mr 42,000 and 44,000) while TG SMP-II is monomeric (Mr 43,000). Western blot analysis revealed that the 42,000 and 44,000-Da proteins cross-react with anti-TG SMP-II antibodies, suggesting that these proteins share common structural properties. The anti-TG SMP-I and Ic antibodies do not cross-react with RU SMP-II and -IV. Likewise, the anti-TG SMP-II antibodies do not cross-react with RU SMP-I and -IV, implying that these enzymes are distinct. RU SMP-IV is composed of a catalytic subunit (Mr 40,000) and a subunit with a molecular weight of 60,000 or 58,000. All three rabbit uterine smooth muscle phosphatases dephosphorylate the isolated myosin light chains but only RU SMP-IV dephosphorylates heavy meromyosin. However, when the catalytic subunit of RU SMP-I is dissociated from the regulatory subunits, it is active toward heavy meromyosin and exhibits higher activity toward myosin light chains and phosphorylase a than its holoenzyme. The substrate specificity of these enzymes and the effects of ATP, NaF, pyrophosphate, okadaic acid, Mg2+, Mn2+, and Ca2+ on their activities are very similar to those of the turkey gizzard smooth muscle phosphatases.     

Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum

Ca2+/calmodulin-dependent protein kinase II, an abundant brain protein proposed to mediate a number of Ca2+-regulated processes in neuronal tissue, is composed of autophosphorylatable subunits of Mr 50,000 and 60,000/58,000. A recent study (McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J.R., and Cohen, P. (1983) FEBS Lett. 163, 329-334) suggested that this kinase exists as isozymes which vary in the relative ratio of these subunits in different tissues or species. Other studies (Walaas, S. I., Nairn, A. C., and Greengard, P. (1983) J. Neurosci. 3, 291-301, 302-311) provided evidence which suggested that the ratio of these phosphopeptides might vary in different brain regions. In the present investigation, we have tested this possibility by comparing Ca2+/calmodulin-dependent protein kinase II purified from rat forebrain and cerebellum. The two kinases had similar purification characteristics, subunit compositions, physical properties, and substrate specificities. Gel filtration and sucrose density gradient centrifugation provided an estimated molecular weight of 550,000 for the forebrain kinase and 615,000 for the cerebellar kinase. The kinases from the two regions clearly differed in the relative proportions of the Mr 50,000 and 60,000/58,000 subunits. Three independent methods indicated that the forebrain kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 3:1 ratio, while the cerebellar kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 1:4 ratio. The forebrain kinase subunits were shown to be identical to the corresponding subunits of the cerebellar kinase by several criteria. The data are consistent with the existence in various brain regions of isozymic forms of Ca2+/calmodulin-dependent protein kinase II which differ in their relative subunit ratios.     

Plasma membrane phosphoproteins in normal and Rous sarcoma virus transformed chick embryo fibroblasts: characterization by in vitro phosphorylation.

Plasma membranes isolated from normal and RSV transformed chick embryo fibroblasts were phosphorylated in vitro using endogenous protein kinase and ATP (gamma32P) and the labeled phosphoproteins were analyzed by SDS-PAGE. A number of protein phosphorylation changes were observed following transformation, however in most cases they were relatively small quantitative differences. The four major changes were in proteins of 47,000, 58,000, 75,000 and 135,000 daltons. Decreased phosphorylation of the 47,000 dalton polypeptide was found in transformed cell membranes but this alteration was shown to be due to differences in cell growth rather than transformation. Increase phosphorylation of the 75,000 dalton protein was at least partially related to virus infection. However, increased phosphorylation of the 58,000 and 135,000 dalton polypeptides were entirely transformation specific.     

Tumor antigens of human ad5 in transformed cells and in cells infected with transformation-defective host-range mutants

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.