Characteristics of rat ceruloplasmin from the serum of animals, which received salts of silver with food

Abiogenic Ag(I) ions have electronic structure, similar to Cu(I) ions and can compete with Cu(I) for binding sites of proteins which transport copper from extracellular media to sites of cuproenzyme formation in the cell. Rodents receiving Ag-salts with food develop extracellular deficiency of copper associated with ceruloplasmin (Cp, the major copper-transporting protein in blood serum of vertebrates). The present work focuses on the studies of biochemical and physicochemical properties of Cp, obtained from blood serum of rats, which received AgCl with food for 4 weeks (Ag-rats). Cp-fractions from blood serum of Ag-rats (Ag-Cp) were obtained by ion-exchange chromatography with stepped gradient of NaCl. Each fraction was tested for oxidase and ferroxidase activities by direct measurement of catalytic activity in the gel, and for specific activity in holo-Cp in oxidation of chromogenic substrate. Molecular mass, electrophoretic mobility and ratio of apo- and holo-forms in Ag-Cp fractions were evaluated by immunoblotting. Ag-Cp samples did not contain products of spontaneous partial proteolytic degradation, characteristic of holo-Cp samples. Fractions of Ag-Cp and holo-Cp (from blood serum of control rats) were compared by optical spectra, tertiary structure, susceptibility to thermal denaturation, and by atomic Cu and Ag content. Ag-Cp contained 1-2% Cp, which is similar by spectral and catalytic properties with holo-Cp. [Ag]:[Cu] ration in Ag-Cp samples was about 4:1. As evidenced by circular dichroism and differential scanning calorimetric studies, the major apo-fraction of Ag-Cp lacked tertiary structure of native Cp and was significantly misfolded, which might explain its resistance to spontaneous partial proteolytic degradation.     

Differences in the catalytic properties of fragments of the ceruloplasmin molecule

Human ceruloplasmin (Cp) molecule is split into fragments by a contaminating protease upon storage of enzyme preparations. These fragments were separated by SDSPAAG electrophoresis and their Mr were estimated. Separate fragments were subjected to immunoelectrophoresis in agarose gel containing rabbit antibodies to human Cp. The immunoprecipitation peaks were then specifically stained to reveal the oxidase activity of the fragments towards o-dianisidine and L-cysteine. All the fragments were able to oxidize the latter, however, only the whole Cp molecule and the two of its largest fragments could oxidize the former. It seems likely that oxidation of L-cysteine does not require the presence of several copper ions constituting the catalytic centre of the blue oxidase (Cp.). contrarily, o-dianisidine seems to be oxidized by the multicopper active site of the enzyme rather than by the autonomously acting singular copper(s). Since o-dianisidine is oxidized by the fragments of Cp lacking the C-terminal polypeptide, which was thought to bind all the coppers of the active centre, it was assumed that some of the latter are bound by amino acids located in another part of the molecule.     

Spectral characteristics of the mechanism of oxidase activity of ceruloplasmin

The absorbance and EPR spectra of type 1 and 2 copper-binding centres which are present in ceruloplasmin (Cp) molecule were shown to disappear upon the reduction of the enzyme by ascorbate under anaerobic conditions. The fluorescence band attributed to type 3 Cu was altered concomitantly. The electron-accepting nitroxyl radical added to reduced Cp restored the absorbance, EPR and fluorescence spectra of the oxidase. Only type 1 and 3 copper ions, as judged by spectral changes, can be reduced by ascorbate and then reoxidized by the nitroxyl radical in the azide-treated Cp. The spectral properties of Cp provided by copper ions of different types change simultaneously and concordantly upon oxidation/reduction. This seems to be caused by cooperative interaction of these ions involved in the electron transfer from the donating substrate to the accepting molecule of the nitroxyl radical (in model studies of oxidase reaction) or oxygen (under natural conditions). The copper ions in the active centre of Cp constitute an intramolecular electron transport chain, which may, at least in vitro, function without one of its links.     

Synthesis and secretion of ceruloplasmin by isolated rat hepatocytes

The synthesis and secretion of ceruloplasmin (Cp) by isolated rat hepatocytes were investigated. Cp released by liver cells appeared to have properties similar to those of the blood-circulating protein, i. e. Mr, oxidase activity, immunological specificity and the peptide set of tryptic fingerprints. The polypeptides with Mr of 130,000, 65,000, 48,000 and 18,000 were revealed in Cp isolated from the incubation medium. These results suggest the susceptibility of the single-chain protein molecule (Mr 130,000) to limited proteolysis which is accomplished by the proteases released from the cells. When fresh serum was added to the incubation medium, the proteolytic degradation of Cp proceeded at a much slower rate, which led to an increase in the content of excreted polypeptides with Mr 130,000. The secretion was strongly diminished by the addition of colchicine to the medium. The time of Cp molecule synthesis on membrane-bound polyribosomes (3.5 min) was determined.     

Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate

The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM.     

Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines

Staphylococcal enterotoxin A (SEA) is a potent polyclonal T cell activator. Its activating effect is entirely dependent upon its binding to accessory cells. Monocytes, B cells, and B lymphomas can bind SEA and support activation of T cells. We have earlier found that Raji cells are particularly efficient as accessory cells for SEA-induced T cell proliferation. In the present investigation we have used this cell line for the isolation and characterization of the membrane molecule to which SEA binds. Flow cytometric analysis of cells dually stained with SEA and anti-HLA-DR mAb showed that the amount of bound SEA was proportional to the HLA-DR expression. Electrophoresis of detergent extracts of Raji cells revealed one distinct SEA-binding band with a Mr of 60 to 65 kDa. This band had the same electrophoretic mobility as the MHC class II molecules. A mAb (G8) with the ability to block SEA binding to Raji cells was established. This mAb was shown to bind to the HLA-DR molecule. Both the G8 mAb and an anti-HLA-DR mAb 9-49 inhibited SEA binding to accessory cells and also inhibited SEA-induced, but not PHA-induced, T cell proliferation and production of IL-2. Immunoprecipitation with specific anti-HLA-DR and anti-HLA-DQ mAb demonstrated that SEA binds to the HLA-DR molecule but not to the HLA-DQ molecule. Binding SEA to Raji cells followed by cross-linking and detergent solubilization of cell membranes, electrophoresis, and Western blotting resulted in two SEA-containing bands corresponding to a Mr of 90 and 105 kDa, respectively. Both these bands also contained the HLA-DR molecule and their appearance could be blocked by preincubation of the Raji cells with the G8 mAb. Collectively the results show that the HLA-DR molecule is the main functional molecule for binding of SEA to accessory cells and that this binding of SEA to HLA-DR is a necessary requirement for SEA-induced T cell activation.     

Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents

Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattus norvegicus), albino mice (Mus musculus), Cp?/? mice, and adult humans (Homo sapiens) to investigate the copper–iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp?/? mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp?/? mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60–90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency.     

Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane.

Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.     

Evidence for two functionally distinct forms of the human Ah receptor

The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 on protein blots of HeLa cell cytosol; two bands were detected at 104 and 106 kDa. The photoaffinity ligand, 2-azido-3-[¹²⁵I]iodo-7,8-dibromodibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hour the cells were UV irradiated. Cytosolic and high salt nuclear preparations were isolated and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the protein to membrane. The AhR was visualized on the membrane, revealing two bands. Alignment of an autoradiogram with the membrane revealed that only the 106 kDa (upper) band was photoaffinity labeled. The nuclear fraction contained only the photoaffinity-labeled 106 kDa form of the AhR. The 104 kDa AhR does not appear to be a proteolytic product of the 106 kDa form. Cyanogen bromide fragmentation revealed that both forms contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these results would indicate that the AhR in human cell lines exists in two distinct forms.     

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.     

Oxidation of orcinol by ceruloplasmin and mixed-ligand copper complexes

Unable to oxidize orcinol (3,5-dihydroxytoluene) under conventional conditions, ceruloplasmin (Cp) catalyzes its oxidation when superoxide radicals are injected into the solution with the aid of a high-voltage generator. The O2-. to oxidized orcinol ratio in solution is close to 2:1. The concentration of hydrogen peroxide, which is the product of the Cp-catalyzed dismutase reaction, is about half that of O2-. No slower than by the native enzyme, orcinol in the presence of O2-. is oxidized by Cp depleted of all its type 2 coppers and partly of type 1 Cu2+. Copper complexes with oxalate and pyrophosphate are able to oxidize orcinol under aerobic conditions, one molecule of oxygen being consumed per each oxidized molecule of orcinol. Both the oxidation of orcinol by Cp and by copper complexes are inhibited by cyanide. Orcinol oxidation seems to be caused by singlet oxygen produced in the Cp-catalyzed dismutase reaction.     

Biotin-conjugated reagents as site-specific probes of membrane protein structure: application to the study of the human erythrocyte hexose transporter

A novel labeling procedure using biotin-conjugated protein-modifying reagents has been employed to study the structure and function of the human erythrocyte hexose transporter. The carbohydrate moiety of the isolated, reconstituted transporter was labeled by using galactose oxidase/biotin hydrazide. Cysteine residues, which are essential for transporter function, were tagged with a biotin-conjugated maleimide. Labeling with this reagent inhibited the binding of cytochalasin B to the transporter. Following sodium dodecyl sulfate-gel electrophoresis, labeling of the transporter and its proteolytic fragments was detected by Western blotting and probing with alkaline phosphatase-conjugated avidin. After tryptic cleavage of the transporter into two membrane domains, preparations reacted with galactose oxidase/biotin hydrazide were labeled on the 25-kDa glycosylated fragment, but not on the carbohydrate-free 19-kDa peptide. Biotin-maleimide-labeled cysteine residues on both peptides. Transporter polypeptide was fragmented more extensively using Staphylococcus aureus V8 protease. Limited digestion produced a broad band of 30-50 kDa and sharper bands of 23 and 21 kDa. More extensive digestion resulted in the disappearance of the 23-kDa peptide and the appearance of sharp bands of 20, 19, 17, 13, 11, 8, and 7 kDa. Biotin label introduced with galactose oxidase/biotin hydrazide was found on the broad 30-kDa band, confirming its identity as a glycopeptide. All of the peptides weighing more than 11 kDa contained cysteine residues labeled with biotin maleimide, while the 8- and 7-kDa peptides were unlabeled. These results demonstrate the potential usefulness of biotin-conjugated reagents as site-specific probes of membrane protein structure.     

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.     

An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture

A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.     

The effect of 110mAg on ceruloplasmin oxidase activity in rats

The effect of single i.v. injection of 110mAgNO3 (0.183 mg Ag+ X kg-1 b.wt.) in rats on the ceruloplasmin oxidase activity (Cp) and copper serum concentration was studied. It was found that Cp activity in the serum decreased to 70% of the control value and simultaneously serum copper concentration decrease to 30% of the control level. In both cases the decrease was independent on the time elapsed after silver administration. Comparing these results with those reported recently in mice Cu deficit in the rat serum was approximately twice higher. This fact is considered to be an inter-species difference. The concentration of copper in the hepatic supernatant significantly decreased (to eight times from control value) after silver injection. Only less than 10% of the total amount of Ag found in whole liver was taken up to hepatic supernatant. GPC analysis of the supernatant (Sephadex G-75) revealed that no Ag-metallothionein fraction is present. On the basis of the results obtained it was concluded that the mechanism of silver inhibition of Cp oxidase activity remains still in question.     

Characterization of an androgen binding protein (ABP) in rat testis and epididymis

An androgen binding protein (ABP) was demonstrated in the 105,000 g supernatant of rat testis homogenate after charcoal extraction of endogenous steroids. Testis ABP proved to be identical to an ABP previously described in rat epididymis. It contained saturable high-affinity sites which exhibited binding specificity for dihydrotestosterone (6) and testosterone when measured by polyacrylamide gel electrophoresis or by competitive binding using charcoal adsorption. Binding to ABP was not affected by ribonuclease or neuraminidase but was decreased by the disulfide reducing agent, dithiothreitol and the sulfhydryl reagent, N-ethylmaleimide. Binding was abolished by treatment with proteolytic enzyme. The mean molecular radius of ABP was 2.92 nm as determined by the retardation of electrophoretic mobility in polyacrylamide gels of decreasing pore size. Assuming a partial specific volume of 0.66–0.74 the molecular weight was 86,000–91,000 for a spherical molecule. ABP binding was stable after treating at 45° C for 20 min. but was destroyed at 60° C. Binding was maximal between pH 7.5 and 9.0 and decreased at pH below 7.0.     

Immunological identification of four different polypeptides in 'subunit VII' of mammalian cytochrome c oxidase

Rat liver cytochrome c oxidase was separated by SDS-gel electrophoresis into 13 polypeptide bands. Monospecific antisera against the isolated polypeptides VIIa, VIIb and VIIc were raised in rabbits. Cytochrome c oxidase was blotted on nitrocellulose and incubated with the antisera. The antisera reacted only with their corresponding polypeptides, indicating no immunological relationship between polypeptides VIIa, VIIb and VIIc. The data also exclude that these polypeptides are proteolytic breakdown products of larger subunits.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Preliminary structural characterization of the leukocyte cell surface molecule recognized by monoclonal antibody TA-1

TA-1 is a monoclonal antibody identifying a cell surface molecule with a broad distribution on normal and malignant human leukocytes. Preliminary structural studies performed by using radioimmunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that TA-1 recognizes a two-chain polypeptide of approximately 170 kilodaltons (KD) and 95 KD under reducing conditions. The same experiment conducted under nonreducing conditions yielded bands of approximately 155 KD and 110 KD, suggesting the existence of intrachain disulfide bonds in both subunits. Both polypeptide chains were labeled with tritiated sodium borohydride after treatment of cells with neuraminidase and galactose oxidase, thereby demonstrating that both were glycosylated. Tryptic peptide mapping indicated that the 170-KD and 95-KD subunits did not have significant homology in peptide composition. We are designating this newly defined human leukocyte bimolecular complex gp 170/95.     

Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase II from the mouse plasmacytoma, MOPC 315.

DNA-dependent RNA polymerase II was purified from the mouse plasmacytoma, MOPC 315. Soluble enzyme was obtained from a nucleoplasmic fraction and subjected to chromatography on phosphocellulose, DEAE-cellulose, and DEAE-Sephadex ion exchange resins and was subjected to sedimentation in sucrose density gradients. A chromatographically homogeneous enzyme was obtained which was purified about 25,000-fold relative to whole cell extracts and which had a specific activity (on native DNA) similar to those reported for other purified eukaryotic class II RNA polymerase preparations. Analysis of purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed three protein bands, designated II-O, II-A, and II-B in order of electrophoretic mobility. The subunit compositions of these nondenatured bands were subsequently analyzed by electrophoresis under denaturing conditions. Each enzyme II form contained subunits with molecular weights of 140,000 (II-c), 41,000 (II-d), 30,000 (II-e), 25,000 (II-f), 22,000 (II-g), 20,000 (II-h), and 16,000 (II-i). Molar ratios were unity for all subunits except subunit II-h which had a molar ratio of 2. Each enzyme form was distinguished by its highest molecular weight subunit. II-O contained subunit II-o (molecular weight 240,000), II-A contained subunit II-a (molecular weight 205,000), and II-B contained subunit II-b (molecular weight 170,000). Total molecular weights for II-O, II-A, and II-B were calculated as 554,000, 519,000, and 484,000, respectively. In addition, the number of RNA polymerase II molecules per MOPC 315 tumor cell was calculated to be about 5 times 10-4.