ARCHIVES
OF
BIOCHEMISTRY
Nicotinamide
AND
BIOPHYSICS
180,
(1977)
Mononucleotide Adenylyltransferase, Chromatin Protein lmmunochemical
WALTER Department
34-40
Comparative
CANTAROW2
of Biochemistry
AND
and Pharmacology, Boston, Massachusetts Received
March
a Nonhistone
Studies’
B. DAVID
STOLLAR3
Tufts University 02111
School
of Medicine,
25, 1976
Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit y globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no crossreactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 M urea, but was reduced by exposure to 6.0 M urea plus 0.4 M guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.
The preceding article (1) described the purification of NMN4 adenylyltransferase (EC 2.7.7.1) from chicken erythrocytes. It has previously been shown that this enzyme is located in the nucleus, both in chick erythrocytes (2) and in other tissues (3). This article describes immunochemical comparisons of the enzyme as isolated from a variety of sources, indicating that the structure of the protein has undergone evolutionary change with species variation, but is very similar in several tissues of one species. Experiments described in this article also explore the association of the enzyme with chromatin.
MATERIALS
AND
METHODS
Materials used in the enzyme purification and assay were as described in the preceding article (1). Chicken liver and pig liver acetone powders were purchased from Sigma Chemical Company. Pigeons were purchased from Connecticut Valley Biological Supply Company (Southampton, Massachusetts), and jumbo bullfrogs were from NASCO (Fort Atkinson, Wisconsin). Cod blood was a gift from the New England Aquarium (Boston Massachusetts). Sephadex G-200 was purchased from Pharmacia Fine Chemicals, hydroxylapapatite as Bio-Rad HTP was from Bio-Rad Laboratories, and complete Freund’s adjuvant was from Difco Laboratories. Preparation of nuclei. Nuclei were isolated from chicken, pheasant, turkey, goose, cod, and frog erythrocytes by a modification of the procedure of Malkin and Denstedt (2) as described in the preceding article (1). Nuclei were isolated from fresh or frozen chicken thymus, kidney, or liver and from pigeon liver by a modification of the method of Chauveau et al. (4). Tissue was homogenized in 0.25 M sucrose-O.003 M MgCl, with a rotating Teflon pestle until the tissue was finely dispersed; usually four to six vertical strokes were required. This homogenate was centri-
I Supported by Grant GB37937 (BMS 73-06883 A02) from the National Science Foundation. 2 Present address: Immunobiology Research Center, University of Wisconsin, Madison, Wisconsin 53706. 3 To whom to address correspondence and reprint requests. 4 Abbreviations used: NMN, nicotinamide mononucleotide; DEAE, diethylaminoethyl. 34 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
EElEN
0003~9s5E
NMN
ADENYLYLTRANSFERASE
IMMUNOCHEMISTRY
fuged at 200g for 10 min to obtain a crude nuclear pellet. The pellet was resuspended in a minimal volume of 0.25 M sucrose-O.003 M MgCl, and layered on top of at least 20 ml of 2.3 M sucrose-O.003 M MgCl,. Nuclei were pelleted through the dense sucrose by centrifugation at 100,OOOg for 1 h. The nuclear pellet was resuspended in homogenizing fluid with vortex mixing and the suspension was stored at 4°C. Enzyme preparations. Soluble NMN adenylyltransferase was prepared from avian erythrocytes through the stage of the 0.3 M NaCl extraction from insoluble chromatin (1). The enzyme was prepared from cod and frog erythrocyte nuclei and from the nuclei of chicken liver, thymus, and kidney and pigeon liver by direct extraction of the nuclei with 1 vol of 0.3 M NaCl, 0.2 M potassium phosphate, pH 7.2. The soluble cod enzyme extract became turbid when incubated at 37°C for 10 min. This turbidity interfered with the enzyme assay; it was removed by preincubating the extract for 3.5 h at 37°C and centrifuging it at 62,000g for 10 min. No additional precipitate formed on further incubation at 37°C for 1 h. Enzyme was extracted from chicken liver and pig liver acetone powders with 0.1 M potassium phosphate buffer, pH 7.4. The pig liver enzyme was extracted and carried through the ammonium sulfate precipitation step as described by Kornberg (5) and was then dialyzed overnight against 10 mM potassium phosphate buffer, pH 7.4. The chicken liver powder (1.0 g) was extracted with 10 ml of the phosphate buffer for 10 min at room temperature. The resulting suspension was centrifuged for 10 min at 12,000g and the supernatant was saved. Precipitate formed in this supernatant on its further incubation at 37°C and interfered with the enzyme assay. As with that in the cod extract, this precipitate was removed by preincubation of the supernatant at 37°C (for 3.5 h) and by its centrifugation C62,OOOg for
1 h); no further precipitate formed in the resulting supernatant, which contained the enzyme activity. The 37°C incubation did not significantly alter the immunoreactivity of the enzyme (Table I). Preparation of chromatin and chromatin proteins. Chromatin was prepared from chicken erythrocytes as described in the preceding article (1). Chromatin proteins were dissociated from chromatin DNA with 6.0 M urea, 0.4 M guanidine HCl, 0.1% P-mercaptoethanol, and 0.1 M sodium phosphate, pH 7 (6). Complexes of DNA with tightly bound nonhistone proteins were prepared as described by Chytil and Spelsberg (7). Chromatin was stirred for 2 h in 3.0 M NaCl, 7.5 M urea, 0.1 M MgCl,, and 15 mM potassium phosphate buffer, pH 6, and then centrifuged at 100,OOOg for 30 h. The pellet of DNA and tightly bound proteins was washed twice with water and resuspended in 2 mM Tris, pH 8.0. Rabbit antisera. Rabbits were immunized with partially purified enzyme, since immunization with the most highly purified preparation was not effective. Rabbit 293 was injected intradermally at several sites on the sides and back, receiving 60 gg of protein from the most active fractions of a Sephadex G-200 column on which a saline-phosphate nuclear extract was fractionated. The protein was emulsified with complete Freund’s adjuvant. A second injection was given similarly 1 week later. On Day 14, 60 pg of the same protein fraction was injected intravenously without adjuvant, and serum was obtained 1 week later. A late serum was obtained 4 months later, 1 week after an intravenous injection of another 60 pg of the same antigen. Rabbit 346 was immunized three times, at lo-day intervals, with 1.0 ml (7.7 mg) of concentrated active fractions from a Sepharose 6B column (1). The enzyme was given intradermally and subcutaneously with complete Freund’s adjuvant. Serum was obtained 1 week after the last injection. After an additional 2 weeks, an intravenous booster of 1.0 ml of
TABLE IMMUNOCHEMICAL Source Chicken Chicken Chicken Chicken
erythrocyte kidney thymus liver
Turkey erythrocyte Goose erythrocyte Pheasant erythrocyte Pigeon liver Frog erythrocyte Cod erythrocyte Pig liver n 0.1-0.3 M NaCl b 0.15 M NaCl-0.1
COMPARISON
I
OF ATP:NMN
ADENYLYLTRANSFERASES Immunoreactivity rum required
of enzyme
buffer
(pH
index (microliters of seto precipitate 0.05 unit)
4 2 2 2 4 6 2 2 45 No precipitation No precipitation No precipitation
Nuclei” Nucleib Nucleib Fresh nuclei* Acetone powder Nuclei” Nuclei” Nuclei” Nucleib Nucleib Nucleib Acetone powder
extract of chromatin. M potassium phosphate
35
7.2) extract
of nuclei.
with with with
50 ~1 50 ~1 50 ~1
36
CANTAROW
AND
enzyme (7.7 mg) was given and serum was obtained 10 days after this injection. Sheep anti-rabbit y-globulin serum was obtained from an animal that received an initial dose of 20 mg of ammonium sulfate-precipitated rabbit y globulin in complete adjuvant at several intradermal sites. A booster of 25 mg of y-globulin in incomplete adjuvant was given 2 weeks later and the animal was bled at weekly intervals. Samples of 0.2 ml of the early sera were able to precipitate all of the reactive antigen in 0.1 ml of normal rabbit serum. Assays for enzyme activity. These were based on the reduction of NAD+ with alcohol dehydrogenase and measurement of the absorbance at 340 nm of the resulting NADH (1). A unit of enzyme activity is the amount capable of producing 1.0 pmol of NAD per hour. Protein was assayed with a microbiuret assay (8). Sephadex G-200 gel filtration. Soluble enzyme was extracted from chicken erythrocyte nuclei with 0.5 M NaCl, 0.1 M potassium phosphate, pH 7.4. The suspension was centrifuged at 12,OOOg for 10 min, and the supernatant was concentrated by its centrifugation through a Centriflo ultrafilter (Amicon). A sample of 6.0 ml of the concentrated extract was applied to a 2.5 x 30-cm column of Sephadex G-200 equilibrated with 50 mM potassium phosphate, pH 7.0. The column was developed with the same buffer and L&ml fractions were collected. Hydroxylapatite chromatography. Bio-Rad HTP was added directly to excess 0.03 M potassium phosphate buffer, pH 7.4, and swirled gently. Fines were removed by repeated decantation and suspension of the gel. When no more fines were visible in the supernatant after the gel sedimented at room temperature for 15 min, the hydroxylapatite was packed into a column (1.2 x 3.3 cm) and washed with 10 ml of the same buffer before the protein was applied. Complement fixation was performed according to the method of Wasserman and Levine (9), with a total volume of 1.4 ml/reaction mixture and a buffer of 0.14 M NaCl, 0.01 M Tris, pH 7.4, with 1.5 mbf Mg2+, 0.5 mM Ca2+, and 0.1% bovine serum albumin. The stated dilutions of antigen or serum refer to solutions before their addition to reaction mixtures, in which a further sixfold dilution occurred. RESULTS
Immunoprecipitation
of Enzyme
In optimal amounts, rabbit antiserum 293 directly precipitated enzyme activity during an incubation of 30 min at 37”C, and enzyme was fully active in the precipitated complexes (Fig. 1). Soluble complexes were formed in far antibody excess (Fig. 1). The more usual soluble complexes of antigen excess were also formed and
STOLLAR
0 00
I 0.1
I 0.2
I 03
ML
SERUM
I I 0.6 0.5
06
FIG. 1. Immunoprecipitation of chicken erythrocyte enzyme by rabbit serum 293. Samples of 0.5 ml of a saline-phosphate (0.15 M NaCl, 0.1 M phosphate, pH 7.4) extract of nuclei were incubated with increasing amounts of antiserum in a total volume of 1.0 ml for 30 min at 37°C. Mixtures were then centrifuged at 4000g for 15 min. Pellets were resuspended in 1.0 ml of 0.09 M potassium phosphate, pH 7. The resuspended pellets (0) and the supernatants (0) were assayed for enzyme activity and compared to a control tube that contained 0.5 ml of enzyme and 0.5 ml of buffer.
precipitation of activity occurred only when more than 30 ~1 of serum was added to the standard amount of enzyme (Fig. 2). Addition of anti-rabbit globulin serum precipitated the antigen excess complexes, so that, with this double-antibody technique, less than 10 ~1 of the anti-enzyme serum was required and precipitation was more complete (Fig. 2). This procedure thus allowed a more precise comparison of enzymes from different sources than did direct precipitation. The reaction was specific, since the combination of normal rabbit serum and anti-rabbit globulin did not precipitate any enzyme activity by trapping. An index of immunoreactivity was defined as the amount of serum required to precipitate 0.05 unit of enzyme activity from a total of 0.15 to 0.35 unit, as determined from serum titrations (Fig. 3). Enzyme from chicken erythrocytes, liver, thymus, and kidney showed very similar immunoreactivity (Table I). The chicken liver enzyme was equally reactive whether it was obtained from fresh or frozen liver through preparation of nuclei or from an acetone powder.
NMN
ADENYLYLTRANSFERASE
/IL SERUM FIG. 2. Comparison
of enzyme precipitation by single CO)- or double CO)-antibody techniques. Duplicate samples of 0.5 ml of 0.3 M NaCl extract of washed chromatin were incubated with varying amounts of antiserum 293 for 2 h at 3’7°C in a total volume of 1.5 ml. One sample was then centrifuged at 4000g for 10 min, and 1.0 ml of the supernatant was assayed for enzyme activity (0). Sheep antirabbit y globulin (0.1 ml) and carrier normal rabbit serum (20 ~1) were added to the second portion. This mixture was incubated for 60 min at 37”C, overnight at 4”C, and then centrifuged; 1.0 ml of the supernatant was assayed (0). In control tubes containing normal rabbit serum and sheep anti-rabbit y globulin, there was no precipitation of enzyme activity.
In comparison of enzymes from various species with the double-antibody technique, the chicken, goose, pheasant, and turkey erythrocyte enzymes cross-reacted very effectively (Table I). Since significant enzyme activity was not obtained from pigeon erythrocytes, and since the erythrocyte and liver enzymes were identical in the chicken, the pigeon enzyme used for the serological comparison was extracted from liver acetone powder. It was much less reactive with the antiserum than were the enzymes from the other birds tested, since 45 ~1 of serum was required for its precipitation as compared to the 2 to 6 ~1 required for the other bird enzymes. Up to 50 ~1 of serum gave no precipitation of the cod, frog, or pig enzyme (Table I). Absorption of Serum for Complement Fixation Studies Since the antisera with antienzyme activity were induced by partially purified enzyme, only the above inhibition studies could be performed with the original sera. Interfering systems had to be removed be-
37
IMMUNOCHEMISTRY
fore a serum could be used for enzymespecific complement fixation reactions. The antibodies in serum 346 were induced with the enzyme taken through all but the last stage of purification. In this last stage, nonenzyme proteins are bound to a DEAE-Sephadex column, while enzyme passes through directly (1). The DEAE-Sephadex column with the bound nonenzyme proteins was then used as an immunoadsorbant to remove corresponding antibodies. A 5-ml sample of serum was dialyzed against the equilibrating buffer (30 mM potassium phosphate, 10 mM EDTA, 1 mM dithiothreitol, pH 7.41, clarified by centrifugation at 12,000g for 10 min, and passed through the column. The serum that emerged from the column still had full antienzyme activity in the doubleantibody assay, but had lost its antibody activity against the major contaminants. This was evident from tests of immunodiffusion in gels and from analysis of column fractions obtained in a different procedure
/.d.
FIG. 3. Comparison
SERUM
of enzymes from varying sources by immunoprecipitation of activity with the double-antibody technique. The enzymes from chicken erythrocytes (O), pheasant erythrocytes (A), turkey erythrocytes CO), pigeon liver (x), and pig liver (A), corresponding to a total of 0.15 to 0.3 unit in each case, were incubated with increasing amounts of antiserum for 1 h at 37°C and then 0.1 ml of sheep anti-rabbit y globulin and 0.02 ml of normal rabbit serum (as carrier) were added. The mixture was further incubated at 37°C for 1 h and at 4°C overnight. The precipitate was removed and the supernatant was assayed for enzyme activity. Control mixtures received normal rabbit serum in place of the antienzyme serum.
38
CANTAROW
of enzyme preparation, in which a salinephosphate extract of chicken erythrocyte nuclei was fractionated on a hydroxylapatite column (Fig. 4). All of the protein was bound at the starting conditions of loading and washing with 0.03 M phosphate buffer, pH 7.4. Proteins were then eluted with a linear gradient of 0.03 to 0.3 M phosphate buffer, pH 7.4. The whole unabsorbed serum reacted predominantly with a large amount of antigen that was eluted early and did not correspond to enzyme activity (Fig. 4). The absorbed serum detected antigen only when enzyme activity occurred and the serologically and catalytically measured profiles were identical (Fig. 4). After overnight dialysis against the complement fixation buffer, 40% of the enzyme activity was lost; complement fixation showed a concomitant loss of about 25% of the serologically reactive protein. Thus, the absorbed serum, which did precipitate enzyme, was also reacting in complement fixation with an antigen that showed properties identical to those of the enzyme. This parallel behavior was also observed in extraction of both active enzyme and reactive antigen from
FRACTION
NUMBER
FIG. 4. Assays of hydroxylapatite column fractions of a saline-phosphate nuclear extract for enzyme activity (X ), complement fixation reactivity with unabsorbed antiserum 346 diluted l/75 (01, or absorbed antiserum 346 diluted l/75 (0). Twenty milliliters of a 0.15 M NaCl, 0.1 M phosphate extract of nuclei was diluted with 180 ml of water to lower the phosphate concentration to less than 30 mM. The diluted enzyme was applied to a lo-ml hydroxylapatite column equilibrated with 30 mM phosphate, pH 7.4, and the column was washed with 50 ml of the equilibrating buffer. Protein was then eluted with a linear potassium phosphate gradient (200 ml of 0.03 M t0 200 Id Of 0.3 M).
AND
STOLLAR
PG/ML
washed absorbed
WA
Micro-complement fixation reaction of intact chicken erythrocyte chromatin with serum ‘346 diluted l/75.
nuclei with 0.1 M phosphate-O.15 M NaCl and from chromatin with 0.3 M NaCl. The absorbed serum reacted with intact isolated chromatin in complement fixation assays (Fig. 5). The reactive component could be identified as a nonhistone chromatin protein according to widely used preparative procedures. It stayed with the chromatin that pelleted during washing in 15 mM sodium phosphate buffer, pH 6.0, but was released into the supernatant when the chromatin was extracted with 3.0 M NaCl, 7.5 M urea, 0.1 mM MgC12, 15 mM potassium phosphate buffer and then centrifuged at 100,OOOg for 30 h; the redissolved pellet in the latter case had less than 1/20th as much reactive antigen as did the supernatant. The absorbed serum also reacted with protein dissociated from chromatin by urea and guanidine in another procedure for preparation of nonhistone proteins (6). Since this procedure destroyed catalytic activity, the stability of the immunoreactive protein was explored further. Partially purified enzyme, at the stage of the 0.3 M NaCl extraction from chromatin, was incubated in various media, diluted with complement fixation buffer, and tested for residual immunoreactivity. Incubation for 15 h at 4°C in 2.0 M NaCl or 2.0 M NaCl with 5.0 M urea, 0.1 mM MgCl*, and 15 mM potassium phosphate buffer, pH 7.0, did not significantly alter reactivity, while a urea-guanidine combination (6.0 M urea, 0.4 M guanidine, 0.1% /3-mercaptoethanol-0.1 M sodium phosphate buffer, pH 7.0) decreased the immunoreactivity by 60% (Fig. 6).
_ . ..” ?--------= ,..’ / __ ,..’ ,f’ I0 80 /I ,..’ I ,I’ / ,..’ ,I z&J .i’ 0LI f’ / ,‘, i” sIL‘IO,i’ ,’ I 2 ..:’ ‘! r ’ ..: : 0.’,..’ NMN
ADENYLYLTRANSFERASE
pG PROTEIN
FIG. 6. Micro-complement
fixation reactions of chicken erythrocyte enzyme (a 0.3 M NaCl extract of washed chromatin) after simple dilution into C fixation buffer (+) or after exposure to 2.0 M NaCl (0); 2.0 M NaCl plus 5.0 M urea, 0.1 mM MgCl, and 15 mM potassium phosphate, pH ‘7 (a); or 6.0 M urea, 0.4 M guanidine, 0.015 M p-mercaptoethanol, 0.1 M sodium phosphate, pH 7 (x). Assays were done with absorbed serum 346 diluted l/75. DISCUSSION
As with many antienzyme systems (10, 111,the antibodies to NMN adenylyltransferase were not directed against the active site, since the enzyme in immune precipitates was fully active. Removal of enzyme activity from solution could, however, be used as a measure of specific reactivity even in the presence of other antigen-antibody systems. The assay was made more efficient by addition of anti-rabbit y-globulin as a second reagent so that even soluble enzyme-antienzyme complexes could be precipitated. The antienzyme activity was not affected by adsorption of other antibodies from the original serum. Comparative study of nuclear proteins is of interest because of varying patterns in their evolution. Some of the histones, for example, have shown very little evolutionary change through all eucaryote evolution (12, 13). While the Hl histone has shown some evolutionary change and even subtypes within a species (14) and the avian erythrocyte H5(F2c) histone is of restricted occurrence (15), the overall pattern is one of conservation of histone structure. This has been taken to indicate that these proteins are not responsible for fine
IMMUNOCHEMISTRY
39
gene regulation. This pattern is also different from that of most enzyme evolution, in which active site structure may be conserved, but other parts of the protein evolve (16). It may mean that all parts of the histone are involved in very specific interactions, with DNA and other histones, that are crucial for normal chromatin structure and function; the histones that are most closely associated with DNA (13) have been the most conserved. Nonhistone proteins show much greater heterogeneity and may include structural, enzymatic, and regulatory proteins (13). Some of these may vary among species and even from tissue to tissue in a way that can be detected immunochemically (7, 17) and functionally (18, 19). It is thus of interest to know whether or not the chromatin enzymes are largely conserved so as to maintain a specific structural relationship to the histones and DNA. The enzyme we studied was active and immunologically very similar in several tissues of one species, but has evolved with species variation in parallel with other proteins. Anti-chicken enzyme antiserum was able to precipitate enzyme from birds of the order Galliformes (chicken, pheasant, and turkey) and the Anseriformes (goose); a similar close relationship was found in a comparison of ovalbumins from these sources (20). The pigeon’s enzyme was quite different from those other bird enzymes, just as was its ovalbumin (20). Enzymes from amphibian, fish, or mammalian sources were not detectably crossreactive with the chicken enzyme. After the serum was absorbed with DEAE-Sephadex, to which the major nonenzyme components were bound, it fully retained its ability to react with enzyme in the double-antibody precipitation assay. To test whether or not the complementfixing antigen was in fact the enzyme, properties of both were compared. As noted, they were both present in the material that passedthrough the DEAE-Sephadex and which, on SDS-polyacrylamide gel electrophoresis, consisted of one major component (1). While this purified enzyme did not enter nondenaturing gels readily, stainable protein that did enter did correspond to enzyme activity (1). Both the en-
40
CANTAROW
AND
zyme and the complement-fixing antigen were separated from a major nonenzyme antigen during a different procedure on elution from hydroxyapatite; the elution profiles of enzyme and antigen were identical. Both enzyme activity and complement-fixing antigen were partially lost on dialysis, possibly by adsorption to the dialysis tubing. The properties of the enzyme and antigen were parallel in these respects and, while it remains possible that a distinct protein was responsible for the complement fixation reaction, it is likely that the enzyme was the reactive component. The complement-fixing antigen was accessible to antibody when chromatin was tested as an antigen; it may be relatively near the surface rather than in close contact with DNA or with more deeply located histones that are less readily accessible to antibody (21). The antigen was dissociated from chromatin by high salt and urea concentrations, but, in this case, it retained serological reactivity after exposure to urea conditions that destroyed catalytic activity. The retention of serological reactivity in this case may relate to the findings of Wakabayashi et al. (171, in which antibodies induced by the protein-DNA complex remaining after treatment of chromatin with high salt and urea concentrations were able to react with the native chromatin. These reagents are often used in the preparation of nonhistone proteins, and it is likely that several of the proteins in such heterogeneous mixtures are, in fact, partially denatured, inactivated enzymes. Antibodies to defined nuclear enzymes should be helpful in attempts to sort out the identities of these proteins, many of which can so far be recognized only as stained bands in electropherograms. REFERENCES 1. CANTAROW,
W., AND STOLLAR,
B. D. (1977)Arch.
STOLLAR
Biochem. Biophys. 180, 26-33. 2. MALKIN, A., AND DENSTEDT, 0. F. (1956) Canad. J. Biochem. PhysioZ. 34, 130-140. 3. SIEBERT, G., AND HUMPHREY, G. B. (1965) in Advances in Enzymology 27, pp. 239-288, Wiley-Interscience, New York. 4. CHAUVEAU, J., MOULB, Y., AND ROUILLER, CH. (1956) Exp. Cell Res. 11, 317-321. 5. KORNBERG, A. (1950) J. Biol. Chem. 132, 779793. 6. LEVY, S., SIMPSON, R., AND SOBER, H. (1972) Biochemistry 11, 1547-1554. 7. CHYTIL, F., AND SPELSBERG, T. (1971) Nature New BioZ. 233, 215-219. 8. ITZHAKI, R., AATD GILL, D. (1964)AnaZ. Biochem. 9, 401-410. 9. WASSERMAN, E., AND LEVINE, L. (1961) J. Zrqmunoz. 87, 290-295. 10. CINADER, B. (1967) in. Antibodies to Biologically Active Molecules (Cinader, B., ed.), pp. l-24, Pergamon, Oxford. R. (1973) in The Antigens (Sela, M., 11. ARNON, ed.), Vol. 1, pp. 88-159, Academic Press, New York. 12. DELANGE, R. J., AND SMITH, E. L. (1975) Ciba Foundation Symp. 28, 59-70. 13. ELGIN, S. C. R., AND WEINTRAUB, H. (1975)Ann. Rev. Biochem. 44, 725-774. 14. BUSTIN, M., AND COLE, R. D. (1968) J. BioZ. Chem. 243,4500-4505. R., AND NEELIN, J. M. (1966) 15. PURKAYASTHA, Biochim. Biophys. Acta 127, 468-477. 16. MARGOLIASH, E., REICHLIN, M., AND NISONOFF, A. (1967) in Conformation of Biopolymers (Ramachandran, G. N., ed.), Vol. 1, p. 253-278, Academic Press, New York. 17. WAKABAYASHI, K., WANG, S., AND HNILICA, L. (1974) Biochemistry 13, 1027-1032. R. S., AFFARA, N., BIRNIE18. PAUL, T., GILMOUR, G., HARRISON, P., HELL, A., HUMPHRIES, S., WINDAS, J., AND YOUNG, B. (1973) Cold Spring Harbor Symp. Quant. BioZ. 38, 885890. T. C., AND KLEIN19 STEIN, G. S., SPELSBERG, SMITH, L. J. (1974) Science 183, 817-824. 20 FEENEY, R., AND ALLISON, R. (1969) Evolutionary Biochemistry of Proteins, Wiley-Interscience, New York. 21. GOLDBLATT, D., AND BUSTIN, M. (1975) Biochemistry 14, 1689-1695.
OF
BIOCHEMISTRY
Nicotinamide
AND
BIOPHYSICS
180,
(1977)
Mononucleotide Adenylyltransferase, Chromatin Protein lmmunochemical
WALTER Department
34-40
Comparative
CANTAROW2
of Biochemistry
AND
and Pharmacology, Boston, Massachusetts Received
March
a Nonhistone
Studies’
B. DAVID
STOLLAR3
Tufts University 02111
School
of Medicine,
25, 1976
Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit y globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no crossreactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 M urea, but was reduced by exposure to 6.0 M urea plus 0.4 M guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.
The preceding article (1) described the purification of NMN4 adenylyltransferase (EC 2.7.7.1) from chicken erythrocytes. It has previously been shown that this enzyme is located in the nucleus, both in chick erythrocytes (2) and in other tissues (3). This article describes immunochemical comparisons of the enzyme as isolated from a variety of sources, indicating that the structure of the protein has undergone evolutionary change with species variation, but is very similar in several tissues of one species. Experiments described in this article also explore the association of the enzyme with chromatin.
MATERIALS
AND
METHODS
Materials used in the enzyme purification and assay were as described in the preceding article (1). Chicken liver and pig liver acetone powders were purchased from Sigma Chemical Company. Pigeons were purchased from Connecticut Valley Biological Supply Company (Southampton, Massachusetts), and jumbo bullfrogs were from NASCO (Fort Atkinson, Wisconsin). Cod blood was a gift from the New England Aquarium (Boston Massachusetts). Sephadex G-200 was purchased from Pharmacia Fine Chemicals, hydroxylapapatite as Bio-Rad HTP was from Bio-Rad Laboratories, and complete Freund’s adjuvant was from Difco Laboratories. Preparation of nuclei. Nuclei were isolated from chicken, pheasant, turkey, goose, cod, and frog erythrocytes by a modification of the procedure of Malkin and Denstedt (2) as described in the preceding article (1). Nuclei were isolated from fresh or frozen chicken thymus, kidney, or liver and from pigeon liver by a modification of the method of Chauveau et al. (4). Tissue was homogenized in 0.25 M sucrose-O.003 M MgCl, with a rotating Teflon pestle until the tissue was finely dispersed; usually four to six vertical strokes were required. This homogenate was centri-
I Supported by Grant GB37937 (BMS 73-06883 A02) from the National Science Foundation. 2 Present address: Immunobiology Research Center, University of Wisconsin, Madison, Wisconsin 53706. 3 To whom to address correspondence and reprint requests. 4 Abbreviations used: NMN, nicotinamide mononucleotide; DEAE, diethylaminoethyl. 34 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
EElEN
0003~9s5E
NMN
ADENYLYLTRANSFERASE
IMMUNOCHEMISTRY
fuged at 200g for 10 min to obtain a crude nuclear pellet. The pellet was resuspended in a minimal volume of 0.25 M sucrose-O.003 M MgCl, and layered on top of at least 20 ml of 2.3 M sucrose-O.003 M MgCl,. Nuclei were pelleted through the dense sucrose by centrifugation at 100,OOOg for 1 h. The nuclear pellet was resuspended in homogenizing fluid with vortex mixing and the suspension was stored at 4°C. Enzyme preparations. Soluble NMN adenylyltransferase was prepared from avian erythrocytes through the stage of the 0.3 M NaCl extraction from insoluble chromatin (1). The enzyme was prepared from cod and frog erythrocyte nuclei and from the nuclei of chicken liver, thymus, and kidney and pigeon liver by direct extraction of the nuclei with 1 vol of 0.3 M NaCl, 0.2 M potassium phosphate, pH 7.2. The soluble cod enzyme extract became turbid when incubated at 37°C for 10 min. This turbidity interfered with the enzyme assay; it was removed by preincubating the extract for 3.5 h at 37°C and centrifuging it at 62,000g for 10 min. No additional precipitate formed on further incubation at 37°C for 1 h. Enzyme was extracted from chicken liver and pig liver acetone powders with 0.1 M potassium phosphate buffer, pH 7.4. The pig liver enzyme was extracted and carried through the ammonium sulfate precipitation step as described by Kornberg (5) and was then dialyzed overnight against 10 mM potassium phosphate buffer, pH 7.4. The chicken liver powder (1.0 g) was extracted with 10 ml of the phosphate buffer for 10 min at room temperature. The resulting suspension was centrifuged for 10 min at 12,000g and the supernatant was saved. Precipitate formed in this supernatant on its further incubation at 37°C and interfered with the enzyme assay. As with that in the cod extract, this precipitate was removed by preincubation of the supernatant at 37°C (for 3.5 h) and by its centrifugation C62,OOOg for
1 h); no further precipitate formed in the resulting supernatant, which contained the enzyme activity. The 37°C incubation did not significantly alter the immunoreactivity of the enzyme (Table I). Preparation of chromatin and chromatin proteins. Chromatin was prepared from chicken erythrocytes as described in the preceding article (1). Chromatin proteins were dissociated from chromatin DNA with 6.0 M urea, 0.4 M guanidine HCl, 0.1% P-mercaptoethanol, and 0.1 M sodium phosphate, pH 7 (6). Complexes of DNA with tightly bound nonhistone proteins were prepared as described by Chytil and Spelsberg (7). Chromatin was stirred for 2 h in 3.0 M NaCl, 7.5 M urea, 0.1 M MgCl,, and 15 mM potassium phosphate buffer, pH 6, and then centrifuged at 100,OOOg for 30 h. The pellet of DNA and tightly bound proteins was washed twice with water and resuspended in 2 mM Tris, pH 8.0. Rabbit antisera. Rabbits were immunized with partially purified enzyme, since immunization with the most highly purified preparation was not effective. Rabbit 293 was injected intradermally at several sites on the sides and back, receiving 60 gg of protein from the most active fractions of a Sephadex G-200 column on which a saline-phosphate nuclear extract was fractionated. The protein was emulsified with complete Freund’s adjuvant. A second injection was given similarly 1 week later. On Day 14, 60 pg of the same protein fraction was injected intravenously without adjuvant, and serum was obtained 1 week later. A late serum was obtained 4 months later, 1 week after an intravenous injection of another 60 pg of the same antigen. Rabbit 346 was immunized three times, at lo-day intervals, with 1.0 ml (7.7 mg) of concentrated active fractions from a Sepharose 6B column (1). The enzyme was given intradermally and subcutaneously with complete Freund’s adjuvant. Serum was obtained 1 week after the last injection. After an additional 2 weeks, an intravenous booster of 1.0 ml of
TABLE IMMUNOCHEMICAL Source Chicken Chicken Chicken Chicken
erythrocyte kidney thymus liver
Turkey erythrocyte Goose erythrocyte Pheasant erythrocyte Pigeon liver Frog erythrocyte Cod erythrocyte Pig liver n 0.1-0.3 M NaCl b 0.15 M NaCl-0.1
COMPARISON
I
OF ATP:NMN
ADENYLYLTRANSFERASES Immunoreactivity rum required
of enzyme
buffer
(pH
index (microliters of seto precipitate 0.05 unit)
4 2 2 2 4 6 2 2 45 No precipitation No precipitation No precipitation
Nuclei” Nucleib Nucleib Fresh nuclei* Acetone powder Nuclei” Nuclei” Nuclei” Nucleib Nucleib Nucleib Acetone powder
extract of chromatin. M potassium phosphate
35
7.2) extract
of nuclei.
with with with
50 ~1 50 ~1 50 ~1
36
CANTAROW
AND
enzyme (7.7 mg) was given and serum was obtained 10 days after this injection. Sheep anti-rabbit y-globulin serum was obtained from an animal that received an initial dose of 20 mg of ammonium sulfate-precipitated rabbit y globulin in complete adjuvant at several intradermal sites. A booster of 25 mg of y-globulin in incomplete adjuvant was given 2 weeks later and the animal was bled at weekly intervals. Samples of 0.2 ml of the early sera were able to precipitate all of the reactive antigen in 0.1 ml of normal rabbit serum. Assays for enzyme activity. These were based on the reduction of NAD+ with alcohol dehydrogenase and measurement of the absorbance at 340 nm of the resulting NADH (1). A unit of enzyme activity is the amount capable of producing 1.0 pmol of NAD per hour. Protein was assayed with a microbiuret assay (8). Sephadex G-200 gel filtration. Soluble enzyme was extracted from chicken erythrocyte nuclei with 0.5 M NaCl, 0.1 M potassium phosphate, pH 7.4. The suspension was centrifuged at 12,OOOg for 10 min, and the supernatant was concentrated by its centrifugation through a Centriflo ultrafilter (Amicon). A sample of 6.0 ml of the concentrated extract was applied to a 2.5 x 30-cm column of Sephadex G-200 equilibrated with 50 mM potassium phosphate, pH 7.0. The column was developed with the same buffer and L&ml fractions were collected. Hydroxylapatite chromatography. Bio-Rad HTP was added directly to excess 0.03 M potassium phosphate buffer, pH 7.4, and swirled gently. Fines were removed by repeated decantation and suspension of the gel. When no more fines were visible in the supernatant after the gel sedimented at room temperature for 15 min, the hydroxylapatite was packed into a column (1.2 x 3.3 cm) and washed with 10 ml of the same buffer before the protein was applied. Complement fixation was performed according to the method of Wasserman and Levine (9), with a total volume of 1.4 ml/reaction mixture and a buffer of 0.14 M NaCl, 0.01 M Tris, pH 7.4, with 1.5 mbf Mg2+, 0.5 mM Ca2+, and 0.1% bovine serum albumin. The stated dilutions of antigen or serum refer to solutions before their addition to reaction mixtures, in which a further sixfold dilution occurred. RESULTS
Immunoprecipitation
of Enzyme
In optimal amounts, rabbit antiserum 293 directly precipitated enzyme activity during an incubation of 30 min at 37”C, and enzyme was fully active in the precipitated complexes (Fig. 1). Soluble complexes were formed in far antibody excess (Fig. 1). The more usual soluble complexes of antigen excess were also formed and
STOLLAR
0 00
I 0.1
I 0.2
I 03
ML
SERUM
I I 0.6 0.5
06
FIG. 1. Immunoprecipitation of chicken erythrocyte enzyme by rabbit serum 293. Samples of 0.5 ml of a saline-phosphate (0.15 M NaCl, 0.1 M phosphate, pH 7.4) extract of nuclei were incubated with increasing amounts of antiserum in a total volume of 1.0 ml for 30 min at 37°C. Mixtures were then centrifuged at 4000g for 15 min. Pellets were resuspended in 1.0 ml of 0.09 M potassium phosphate, pH 7. The resuspended pellets (0) and the supernatants (0) were assayed for enzyme activity and compared to a control tube that contained 0.5 ml of enzyme and 0.5 ml of buffer.
precipitation of activity occurred only when more than 30 ~1 of serum was added to the standard amount of enzyme (Fig. 2). Addition of anti-rabbit globulin serum precipitated the antigen excess complexes, so that, with this double-antibody technique, less than 10 ~1 of the anti-enzyme serum was required and precipitation was more complete (Fig. 2). This procedure thus allowed a more precise comparison of enzymes from different sources than did direct precipitation. The reaction was specific, since the combination of normal rabbit serum and anti-rabbit globulin did not precipitate any enzyme activity by trapping. An index of immunoreactivity was defined as the amount of serum required to precipitate 0.05 unit of enzyme activity from a total of 0.15 to 0.35 unit, as determined from serum titrations (Fig. 3). Enzyme from chicken erythrocytes, liver, thymus, and kidney showed very similar immunoreactivity (Table I). The chicken liver enzyme was equally reactive whether it was obtained from fresh or frozen liver through preparation of nuclei or from an acetone powder.
NMN
ADENYLYLTRANSFERASE
/IL SERUM FIG. 2. Comparison
of enzyme precipitation by single CO)- or double CO)-antibody techniques. Duplicate samples of 0.5 ml of 0.3 M NaCl extract of washed chromatin were incubated with varying amounts of antiserum 293 for 2 h at 3’7°C in a total volume of 1.5 ml. One sample was then centrifuged at 4000g for 10 min, and 1.0 ml of the supernatant was assayed for enzyme activity (0). Sheep antirabbit y globulin (0.1 ml) and carrier normal rabbit serum (20 ~1) were added to the second portion. This mixture was incubated for 60 min at 37”C, overnight at 4”C, and then centrifuged; 1.0 ml of the supernatant was assayed (0). In control tubes containing normal rabbit serum and sheep anti-rabbit y globulin, there was no precipitation of enzyme activity.
In comparison of enzymes from various species with the double-antibody technique, the chicken, goose, pheasant, and turkey erythrocyte enzymes cross-reacted very effectively (Table I). Since significant enzyme activity was not obtained from pigeon erythrocytes, and since the erythrocyte and liver enzymes were identical in the chicken, the pigeon enzyme used for the serological comparison was extracted from liver acetone powder. It was much less reactive with the antiserum than were the enzymes from the other birds tested, since 45 ~1 of serum was required for its precipitation as compared to the 2 to 6 ~1 required for the other bird enzymes. Up to 50 ~1 of serum gave no precipitation of the cod, frog, or pig enzyme (Table I). Absorption of Serum for Complement Fixation Studies Since the antisera with antienzyme activity were induced by partially purified enzyme, only the above inhibition studies could be performed with the original sera. Interfering systems had to be removed be-
37
IMMUNOCHEMISTRY
fore a serum could be used for enzymespecific complement fixation reactions. The antibodies in serum 346 were induced with the enzyme taken through all but the last stage of purification. In this last stage, nonenzyme proteins are bound to a DEAE-Sephadex column, while enzyme passes through directly (1). The DEAE-Sephadex column with the bound nonenzyme proteins was then used as an immunoadsorbant to remove corresponding antibodies. A 5-ml sample of serum was dialyzed against the equilibrating buffer (30 mM potassium phosphate, 10 mM EDTA, 1 mM dithiothreitol, pH 7.41, clarified by centrifugation at 12,000g for 10 min, and passed through the column. The serum that emerged from the column still had full antienzyme activity in the doubleantibody assay, but had lost its antibody activity against the major contaminants. This was evident from tests of immunodiffusion in gels and from analysis of column fractions obtained in a different procedure
/.d.
FIG. 3. Comparison
SERUM
of enzymes from varying sources by immunoprecipitation of activity with the double-antibody technique. The enzymes from chicken erythrocytes (O), pheasant erythrocytes (A), turkey erythrocytes CO), pigeon liver (x), and pig liver (A), corresponding to a total of 0.15 to 0.3 unit in each case, were incubated with increasing amounts of antiserum for 1 h at 37°C and then 0.1 ml of sheep anti-rabbit y globulin and 0.02 ml of normal rabbit serum (as carrier) were added. The mixture was further incubated at 37°C for 1 h and at 4°C overnight. The precipitate was removed and the supernatant was assayed for enzyme activity. Control mixtures received normal rabbit serum in place of the antienzyme serum.
38
CANTAROW
of enzyme preparation, in which a salinephosphate extract of chicken erythrocyte nuclei was fractionated on a hydroxylapatite column (Fig. 4). All of the protein was bound at the starting conditions of loading and washing with 0.03 M phosphate buffer, pH 7.4. Proteins were then eluted with a linear gradient of 0.03 to 0.3 M phosphate buffer, pH 7.4. The whole unabsorbed serum reacted predominantly with a large amount of antigen that was eluted early and did not correspond to enzyme activity (Fig. 4). The absorbed serum detected antigen only when enzyme activity occurred and the serologically and catalytically measured profiles were identical (Fig. 4). After overnight dialysis against the complement fixation buffer, 40% of the enzyme activity was lost; complement fixation showed a concomitant loss of about 25% of the serologically reactive protein. Thus, the absorbed serum, which did precipitate enzyme, was also reacting in complement fixation with an antigen that showed properties identical to those of the enzyme. This parallel behavior was also observed in extraction of both active enzyme and reactive antigen from
FRACTION
NUMBER
FIG. 4. Assays of hydroxylapatite column fractions of a saline-phosphate nuclear extract for enzyme activity (X ), complement fixation reactivity with unabsorbed antiserum 346 diluted l/75 (01, or absorbed antiserum 346 diluted l/75 (0). Twenty milliliters of a 0.15 M NaCl, 0.1 M phosphate extract of nuclei was diluted with 180 ml of water to lower the phosphate concentration to less than 30 mM. The diluted enzyme was applied to a lo-ml hydroxylapatite column equilibrated with 30 mM phosphate, pH 7.4, and the column was washed with 50 ml of the equilibrating buffer. Protein was then eluted with a linear potassium phosphate gradient (200 ml of 0.03 M t0 200 Id Of 0.3 M).
AND
STOLLAR
PG/ML
washed absorbed
WA
Micro-complement fixation reaction of intact chicken erythrocyte chromatin with serum ‘346 diluted l/75.
nuclei with 0.1 M phosphate-O.15 M NaCl and from chromatin with 0.3 M NaCl. The absorbed serum reacted with intact isolated chromatin in complement fixation assays (Fig. 5). The reactive component could be identified as a nonhistone chromatin protein according to widely used preparative procedures. It stayed with the chromatin that pelleted during washing in 15 mM sodium phosphate buffer, pH 6.0, but was released into the supernatant when the chromatin was extracted with 3.0 M NaCl, 7.5 M urea, 0.1 mM MgC12, 15 mM potassium phosphate buffer and then centrifuged at 100,OOOg for 30 h; the redissolved pellet in the latter case had less than 1/20th as much reactive antigen as did the supernatant. The absorbed serum also reacted with protein dissociated from chromatin by urea and guanidine in another procedure for preparation of nonhistone proteins (6). Since this procedure destroyed catalytic activity, the stability of the immunoreactive protein was explored further. Partially purified enzyme, at the stage of the 0.3 M NaCl extraction from chromatin, was incubated in various media, diluted with complement fixation buffer, and tested for residual immunoreactivity. Incubation for 15 h at 4°C in 2.0 M NaCl or 2.0 M NaCl with 5.0 M urea, 0.1 mM MgCl*, and 15 mM potassium phosphate buffer, pH 7.0, did not significantly alter reactivity, while a urea-guanidine combination (6.0 M urea, 0.4 M guanidine, 0.1% /3-mercaptoethanol-0.1 M sodium phosphate buffer, pH 7.0) decreased the immunoreactivity by 60% (Fig. 6).
_ . ..” ?--------= ,..’ / __ ,..’ ,f’ I0 80 /I ,..’ I ,I’ / ,..’ ,I z&J .i’ 0LI f’ / ,‘, i” sIL‘IO,i’ ,’ I 2 ..:’ ‘! r ’ ..: : 0.’,..’ NMN
ADENYLYLTRANSFERASE
pG PROTEIN
FIG. 6. Micro-complement
fixation reactions of chicken erythrocyte enzyme (a 0.3 M NaCl extract of washed chromatin) after simple dilution into C fixation buffer (+) or after exposure to 2.0 M NaCl (0); 2.0 M NaCl plus 5.0 M urea, 0.1 mM MgCl, and 15 mM potassium phosphate, pH ‘7 (a); or 6.0 M urea, 0.4 M guanidine, 0.015 M p-mercaptoethanol, 0.1 M sodium phosphate, pH 7 (x). Assays were done with absorbed serum 346 diluted l/75. DISCUSSION
As with many antienzyme systems (10, 111,the antibodies to NMN adenylyltransferase were not directed against the active site, since the enzyme in immune precipitates was fully active. Removal of enzyme activity from solution could, however, be used as a measure of specific reactivity even in the presence of other antigen-antibody systems. The assay was made more efficient by addition of anti-rabbit y-globulin as a second reagent so that even soluble enzyme-antienzyme complexes could be precipitated. The antienzyme activity was not affected by adsorption of other antibodies from the original serum. Comparative study of nuclear proteins is of interest because of varying patterns in their evolution. Some of the histones, for example, have shown very little evolutionary change through all eucaryote evolution (12, 13). While the Hl histone has shown some evolutionary change and even subtypes within a species (14) and the avian erythrocyte H5(F2c) histone is of restricted occurrence (15), the overall pattern is one of conservation of histone structure. This has been taken to indicate that these proteins are not responsible for fine
IMMUNOCHEMISTRY
39
gene regulation. This pattern is also different from that of most enzyme evolution, in which active site structure may be conserved, but other parts of the protein evolve (16). It may mean that all parts of the histone are involved in very specific interactions, with DNA and other histones, that are crucial for normal chromatin structure and function; the histones that are most closely associated with DNA (13) have been the most conserved. Nonhistone proteins show much greater heterogeneity and may include structural, enzymatic, and regulatory proteins (13). Some of these may vary among species and even from tissue to tissue in a way that can be detected immunochemically (7, 17) and functionally (18, 19). It is thus of interest to know whether or not the chromatin enzymes are largely conserved so as to maintain a specific structural relationship to the histones and DNA. The enzyme we studied was active and immunologically very similar in several tissues of one species, but has evolved with species variation in parallel with other proteins. Anti-chicken enzyme antiserum was able to precipitate enzyme from birds of the order Galliformes (chicken, pheasant, and turkey) and the Anseriformes (goose); a similar close relationship was found in a comparison of ovalbumins from these sources (20). The pigeon’s enzyme was quite different from those other bird enzymes, just as was its ovalbumin (20). Enzymes from amphibian, fish, or mammalian sources were not detectably crossreactive with the chicken enzyme. After the serum was absorbed with DEAE-Sephadex, to which the major nonenzyme components were bound, it fully retained its ability to react with enzyme in the double-antibody precipitation assay. To test whether or not the complementfixing antigen was in fact the enzyme, properties of both were compared. As noted, they were both present in the material that passedthrough the DEAE-Sephadex and which, on SDS-polyacrylamide gel electrophoresis, consisted of one major component (1). While this purified enzyme did not enter nondenaturing gels readily, stainable protein that did enter did correspond to enzyme activity (1). Both the en-
40
CANTAROW
AND
zyme and the complement-fixing antigen were separated from a major nonenzyme antigen during a different procedure on elution from hydroxyapatite; the elution profiles of enzyme and antigen were identical. Both enzyme activity and complement-fixing antigen were partially lost on dialysis, possibly by adsorption to the dialysis tubing. The properties of the enzyme and antigen were parallel in these respects and, while it remains possible that a distinct protein was responsible for the complement fixation reaction, it is likely that the enzyme was the reactive component. The complement-fixing antigen was accessible to antibody when chromatin was tested as an antigen; it may be relatively near the surface rather than in close contact with DNA or with more deeply located histones that are less readily accessible to antibody (21). The antigen was dissociated from chromatin by high salt and urea concentrations, but, in this case, it retained serological reactivity after exposure to urea conditions that destroyed catalytic activity. The retention of serological reactivity in this case may relate to the findings of Wakabayashi et al. (171, in which antibodies induced by the protein-DNA complex remaining after treatment of chromatin with high salt and urea concentrations were able to react with the native chromatin. These reagents are often used in the preparation of nonhistone proteins, and it is likely that several of the proteins in such heterogeneous mixtures are, in fact, partially denatured, inactivated enzymes. Antibodies to defined nuclear enzymes should be helpful in attempts to sort out the identities of these proteins, many of which can so far be recognized only as stained bands in electropherograms. REFERENCES 1. CANTAROW,
W., AND STOLLAR,
B. D. (1977)Arch.
STOLLAR
Biochem. Biophys. 180, 26-33. 2. MALKIN, A., AND DENSTEDT, 0. F. (1956) Canad. J. Biochem. PhysioZ. 34, 130-140. 3. SIEBERT, G., AND HUMPHREY, G. B. (1965) in Advances in Enzymology 27, pp. 239-288, Wiley-Interscience, New York. 4. CHAUVEAU, J., MOULB, Y., AND ROUILLER, CH. (1956) Exp. Cell Res. 11, 317-321. 5. KORNBERG, A. (1950) J. Biol. Chem. 132, 779793. 6. LEVY, S., SIMPSON, R., AND SOBER, H. (1972) Biochemistry 11, 1547-1554. 7. CHYTIL, F., AND SPELSBERG, T. (1971) Nature New BioZ. 233, 215-219. 8. ITZHAKI, R., AATD GILL, D. (1964)AnaZ. Biochem. 9, 401-410. 9. WASSERMAN, E., AND LEVINE, L. (1961) J. Zrqmunoz. 87, 290-295. 10. CINADER, B. (1967) in. Antibodies to Biologically Active Molecules (Cinader, B., ed.), pp. l-24, Pergamon, Oxford. R. (1973) in The Antigens (Sela, M., 11. ARNON, ed.), Vol. 1, pp. 88-159, Academic Press, New York. 12. DELANGE, R. J., AND SMITH, E. L. (1975) Ciba Foundation Symp. 28, 59-70. 13. ELGIN, S. C. R., AND WEINTRAUB, H. (1975)Ann. Rev. Biochem. 44, 725-774. 14. BUSTIN, M., AND COLE, R. D. (1968) J. BioZ. Chem. 243,4500-4505. R., AND NEELIN, J. M. (1966) 15. PURKAYASTHA, Biochim. Biophys. Acta 127, 468-477. 16. MARGOLIASH, E., REICHLIN, M., AND NISONOFF, A. (1967) in Conformation of Biopolymers (Ramachandran, G. N., ed.), Vol. 1, p. 253-278, Academic Press, New York. 17. WAKABAYASHI, K., WANG, S., AND HNILICA, L. (1974) Biochemistry 13, 1027-1032. R. S., AFFARA, N., BIRNIE18. PAUL, T., GILMOUR, G., HARRISON, P., HELL, A., HUMPHRIES, S., WINDAS, J., AND YOUNG, B. (1973) Cold Spring Harbor Symp. Quant. BioZ. 38, 885890. T. C., AND KLEIN19 STEIN, G. S., SPELSBERG, SMITH, L. J. (1974) Science 183, 817-824. 20 FEENEY, R., AND ALLISON, R. (1969) Evolutionary Biochemistry of Proteins, Wiley-Interscience, New York. 21. GOLDBLATT, D., AND BUSTIN, M. (1975) Biochemistry 14, 1689-1695.
