Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel

A monoclonal antibody, mAb 1A, that immunoprecipitates the [3H]PN200-110-binding complex from rabbit skeletal muscle has been used to study the subunit structure of the dihydropyridine-sensitive, voltage-activated calcium channel. Digitonin-solubilized [3H]PN200-110-binding component, purified by wheat germ agglutinin chromatography, sediments as a 21 S complex. The sedimentation coefficient of the complex is increased to about 24 S after incubation with mAb 1A IgG. Four polypeptides with apparent molecular weights under nonreducing conditions of 220,000, 200,000, 61,000, and 33,000 co-sediment with the 21 S complex. mAb 1A recognizes the Mr 200,000 polypeptide, as shown by Western blotting analysis. [3H] PN200-110 complex purified by wheat germ agglutinin chromatography followed by immunoaffinity chromatography on an mAb 1A column is comprised primarily of the same four polypeptides. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the Mr 220,000 protein migrates as a polypeptide of Mr 143,000; the mobility of the Mr 200,000 protein recognized by mAb 1A is unaffected by reduction. Thus, the Mr 200,000 polypeptide appears to be a previously undescribed component of the dihydropyridine-binding complex and, in association with the other polypeptides, may comprise the voltage-sensitive calcium channel.     

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.     

Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form

Three polypeptides with apparent Mr = 200,000, 135,000, and 115,000, reacting with antibody to human ceruloplasmin (Cp), were consistently found in sera of normal adult and newborn subjects, patients with Wilson's disease, as well as in the oxidase-active fraction of purified human Cp, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentrations of the three Cp polypeptides were proportional to the total Cp oxidase activity measured in whole serum. Peptide mapping revealed that the three Cp polypeptides were closely related. Cross-linking of Cp135 resulted in dimers with electrophoretic mobility similar to that of Cp200. A common shift in electrophoretic mobility following N-glycanase treatment indicated that all three polypeptides were N-glycosylated, and that the apparent differences in molecular mass could not be related to the carbohydrate moiety. Immunoprecipitates of cell lysates of [35S]cysteine labeled HepG2 cells revealed the presence of two species of newly synthesized Cp polypeptides, Mr 200,000 and 135,000, which were secreted into the media. Secretion of Cp200 by the human liver appears to be physiologic and may be the result of posttranslational modification of Cp135.     

Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels.

A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000.     

Structure of proteoheparan sulfates from fibroblasts. Confluent and proliferating fibroblasts produce at least three types of proteoheparan sulfates with functionally different core proteins

[3H]Leucine- and [35S]sulfate-labeled proteoheparan sulfates were isolated from postconfluent or proliferating cultures of human skin fibroblasts. Cell layers were solubilized by Triton X-100, and transferrin-binding macromolecules were isolated by affinity chromatography. Proteoglycans with no affinity for transferrin were purified by using ion-exchange and gel permeation chromatography. Postconfluent cells synthesize a proteoheparan sulfate of Mr 350,000 (as determined by gel permeation chromatography) which has affinity for transferrin as well as for octyl-Sepharose. Its core protein (Mr 180,000) consists of two disulfide-bonded polypeptides of Mr 90,000. This species was not detected in cultures of proliferating cells. Proliferating and confluent cells also synthesize other forms of proteoheparan sulfates (Mr 200,000-400,000) which have no affinity for transferrin. However, most of them have affinity for octyl-Sepharose. The core protein of proteoheparan sulfates made by proliferating cells has Mr 50,000. A smaller form (Mr 250,000) of this proteoglycan was solubilized by Triton X-100, whereas a larger form (Mr 400,000) remained associated with the pericellular matrix. A third type of proteoheparan sulfate (Mr 200,000) without affinity for transferrin nor octyl-Sepharose was associated with postconfluent cell layers but not with proliferating ones. Its core protein has Mr 35,000. Heparan sulfate oligosaccharides (Mr 6,000 or higher) were found in proliferating cells but not in postconfluent ones.     

Isolation and partial characterization of an Mr 60,000 subunit of a type 2A phosphatase from rabbit reticulocytes

A Mr 60,000 peptide that modulates the activity of the Mr 35,000 catalytic subunit of a type 2A phosphatase has been isolated from rabbit reticulocytes and partially characterized. The peptide appears to be a subunit of the intact phosphatase that has been isolated under nondenaturing conditions. The Mr 60,000 peptide itself is catalytically inactive. However, it binds to the Mr 35,000 catalytic subunit causing a decrease in its activity for dephosphorylation of phosphorylated 40 S ribosomal subunits, but an increase in dephosphorylation of peptide initiation factor 2 phosphorylated in its alpha subunit. Reassociation of the Mr 60,000 and the Mr 35,000 peptides yields a two-subunit phosphatase with a Stokes radius of 42 A; sedimentation coefficient, S20,w of 5.1 S; molecular weight of 89,000. These parameters are compared to those of the native three-subunit enzyme and those of the isolated Mr 35,000 and 60,000 peptides.     

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Sch?gger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.     

Biosynthesis of mammalian DNA ligase

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.     

Identification of a novel Mr = 76-kDa form of beta-adrenergic receptors

The structure of the human beta-adrenergic receptor in purified basal membranes of human placental syncytiotrophoblast was probed using photoaffinity labeling. Basal membranes display a high specific activity of receptors (4-5 pmol/mg protein) and possess both beta 1- and beta 2-adrenergic receptors subtypes. Autoradiography of membranes that were incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, identified four radiolabeled peptides, Mr = 65-kDa, 54-kDa, 43-kDa and a novel higher molecular weight 76-kDa form of the receptor. Photoaffinity labeling of each of these four peptides displayed the pharmacological properties expected for true beta-adrenergic receptors. The 76-kDa photoaffinity labeled receptor peptide observed in human placenta basal membranes has not been reported elsewhere. Competition studies with the beta1-selective ligand CGP-20712A demonstrate that the photoaffinity labeled receptor peptides are composed of both beta 1- and beta 2-adrenergic receptor subtypes.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

The isolation and properties of multiple forms of folate binding protein in cultured KB cells

The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.     

Purification and properties of human platelet P235. A high molecular weight protein substrate of endogenous calcium-activated protease(s)

Two major high molecular weight proteins of human platelets are highly susceptible to proteolytic degradation by endogenous calcium-activated protease activities. Of the two proteins, one has been identified as filamin (Mr = 250,000 subunit); the second, a Mr = 235,000 subunit protein contributing 3-8% of the total platelet proteins, has not been previously characterized. We have now purified this protein, designated P235, to apparent homogeneity (greater than 95%). P235 was extracted by a Triton X-100 and EDTA containing buffer at pH 9.0 and purified by a series of DEAE-cellulose, phosphocellulose, and gel filtration chromatographies. Purified P235 is a dimer of Mr = 235,000 subunit. Its Stokes radius (67 A) and frictional ratio of 1.3 suggest that P235 is approximately globular. Despite this similarity in subunit and molecular weight of P235 to filamin, spectrin, fibronectin, and myosin, its amino acid composition, immunological properties, and peptide map are distinctly different and showed no precursor-product relationship to these proteins. Calcium-activated protease(s) in crude platelet extract rapidly degrade P235 into a Mr = 200,000 stable fragment. Upon prolonged storage at 4 degrees C, purified P235 partially degrades into a Mr = 220,000 and a Mr = 200,000 fragment. This degradation pattern suggests that P235 contains a large Mr = 200,000 protease-resistant domain. The availability of pure P235 will be useful in elucidating the functional role of this platelet protein, as well as the role of calcium-activated proteases in platelet function.     

Studies on the primary structure of rat liver phenylalanine hydroxylase

The primary structure of phenylalanine hydroxylase purified from rat liver was investigated with high speed gel filtration chromatography, cyanogen bromide cleavage and end group analyses of polypeptides derived from the enzyme. On gel filtration in the presence of 6M guanidine hydrochloride, the enzyme gave a single peak corresponding to a molecular weight of 52,000. In the same system the enzyme that had been cleaved with cyanogen bromide gave two peptides (CB1, Mr = 32,800 and CB2, Mr = 20,400). Sequence studies showed that the alignment of these two peptides was CB1 - CB2. Furthermore, in experiments using 32P phosphorylated enzyme, the site of phosphorylation by cAMP-dependent protein kinase was found to be located on the CB1 peptide. The NH2-terminus of this enzyme, which was found to be blocked, was shown to be N-acetylalanine. By both carboxypeptidase A digestion and hydrazinolysis, the carboxyl terminus was identified as serine. These data indicate that the phenylalanine hydroxylase molecule from rat liver is composed of subunits which are homogenous or, at least, very similar in their primary structure.     

Structure and proteolysis of the growth hormone receptor on rat hepatocytes

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.     

Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain

Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.     

Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers

Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

Synthesis of high molecular weight thyroglobulin peptides by thyroid polysome in vitro.

Bovine thyroid polysomes were isolated under conditions which had yielded large polysomes in other systems. Between 25 and 40% of the protein synthesized by these polysomes could be precipitated by thyroglobulin antibody. When these immunoprecipitates were separated by dodecyl sulfate-polyacrylamide gel electrophoresis (4% running gel), over 50% of the radioactivity was located in the regions of polypeptides greater than 100,000 daltons. Between 11 and 13% of the total radioactivity was found as a single peak co-migrating with the main band of bovine thyroglobulin (Mr = 330,000). Peaks of radioactivity were also found in regions of molecular weights between 130,000 and 200,000. When the immunoprecipitates were separated in a 10% running gel, about 50% of the radioactivity was located in the top 8 mm of the gel. Most of the remaining radioactivity was distributed in regions corresponding to molecular weights greater than 68,000. No peak of radioactivity was seen corresponding to peptides of 15,000 daltons.