THE .JOURNAL OF BIOLOGICAL CHE~STRY Vol. 243, No.10, Issue of May 25, pp. 2507-2513, 1968

I+ktea in U.S. A.

Characterization and Immunochemical Localization of a Basic Protein from Pig Brain

II. PEPTIDE MAPS AND TISSUE-SPECIFIC NUCLEAR LOCALIZATION*

(Received for publication, October 19, 1967)

LAWRENCE G. TOWIASI AXD S. E. KORNGUTH

From the Departments of Neurology and Physiological Chemistry, University of Wisconsin Medical Center, Madison, Wisconsin 53706

SUMMARY

The procedure for the purification of a basic protein has been reported. Its homogeneity has been established by ultracentrifugation, starch gel electrophoresis, polyacrylamide gel electrophoresis, and end group analysis.

Peptide maps of complete tryptic hydrolysates indicate that the molecular weight of this basic protein is 27,000. By immunohistological techniques, it was shown that fluorescent antibody, prepared against the basic protein, reacted specifi- cally with the nuclei of neurons and spermatogonia and did not react with the cell populations in kidney, liver, ovary, and spleen of the same animal. An identical tissue specificity was observed in a variety of animals from tadpole to monkey.

In recent years, the hypothesis that the histones are involved in the regulation of cellular function (l-8) has stimulated con- siderable interest in the characterization of these proteins. Be- cause it seems most probable that many of the histone fractions studied thus far are heterogeneous (9), the number of distinct histones is unknown (9, 10). There is little agreement upon the existence of an organ-specific histone. Stedman has provided evidence that the arginine-rich P-h&ones from the ox (11) and the domestic fowl (12) are tissue- as well as species-specific. Neelin and Butler (13) showed that 18 zones were resolved on electrophoresis of the histones isolated from chickens, and that Zone 15 was characteristic of chicken erythrocytes. On the other hand, the two main fractions of histones prepared from ,calf thymus, liver, and kidney and from guinea pig testis were nearly identical in t,heir amino acid composition (14). In addi- tion, the chromatographic patterns of the peptides released by

* This work was supported by Grant NB05-631 from the Na- tional Institute of Ne;Iological-diseases, and Blindness and by Grant GM-01666 from the National Institutes of Health. The workwas carried out during t.he t.enure of a Life Insurance Medical Research Fund Fellowship (I,. G. T.).

tryptic digestion were also similar (14). Piha, C&nod, and Waelsch (15) and Neidle and Waelsch (16) have concluded from the identical electrophoretic patterns of histone fractions isolated from brain, liver, and kidney that, as with DNA, the over-all composition of histones is not organ-specific. Similar conclu- sions on the lack of organ specificity in histones were arrived at by Hnilica, Johns, and Butler (17) on the basis of amino acid composition and amino-terminal groups of histone fractions from the thymus, spleen, and liver of calves. Other groups (18-20) agree that the primary structures of the histones isolated from different organs are identical. They conclude that the methylation of the e-amino groups of lysine (19, 21, 22) and the phosphorylation of the serine and threonine residues (5, 18, 20, 23), that are detected in a small percentage of the histones, could account for their postulated specificity.

Immunological techniques have only recently been used to investigate histone specificity. This appears to be due to the fact that histones were thought to be either weak antigens or antigenic (24, 25). Rtimke and Sluyser (26), however, recently showed the antigenicity of the lysine-rich histones.

Immunohistological procedures (27) have been used to de- termine t.he intracellular localization of many specific macro- molecules such as viruses (28) and procarboxypeptidases (29). This localization procedure depends upon the reaction of a modified antibody with a specific macromolecule (antigen) in situ. The recent application of fluorescent antibody techniques to the localization of a low molecular weight basic protein within the myelin sheath (30, 31) and the lysine-rich histones from liver within the liver cell nucleus (26) illustrates the possibility that this technique may be used to determine whether organ-specific and tissue-specific histones exist.

A procedure has been reported (32) for the preparation of large quantities of a basic protein from pig brain. The homo- geneity of this basic protein has been established by ultracentrifu- gation, starch gel electrophoresis, polyacrylamide gel electro- phoresis, and end group analysis (32). Many of the properties of the protein (32) are similar to those of the histones (9, 10,33).

In the present report, the pig brain basic protein was further characterized by peptide mapping after tryptic hydrolysis. The

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Characterization and Immunochemical Localization of Basic Protein from Pig Brain. II Vol. 243, No. 10

localization of the pig brain basic protein within the nervous system and the presence of antigenically similar proteins in testes were determined with the aid of fluorescent immuno- histochemical methods.

EXPERIMENTAL PROCEDURE

MaterialsTrypsin (twice crystallized) was purchased from Worthington. Ninhydrin and isatin were obtained from Sar- gent (Chicago, Illinois). Ionagar was purchased from Con- solidated Laboratories (Chicago, Illinois). The Paragon mul- tiple stain was purchased from Paragon C and C (New York, New York) and the fluorescein isothiocyanate was obtained from Nutritional Biochemicals.

Tryptic Digestion-The method used to follow the course and extent of tryptic digestion was a modification of the procedure described by Hirs, Moore, and Stein (34). Twenty milligrams of the basic protein were incubated at 37” with trypsin (1% by weight of substrate) in 2 ml of sodium bicarbonate buffer (0.25 M, pH 8.0) containing 0.001 M CaC12. Samples of the incubation mixture were taken at 0.5, 1, 3, 5, 10, and 20 hours. The rela- tive increase in ninhydrin-positive material in these samples was determined by the procedure of Moore and Stein (35).

For peptide mapping, 100 mg of the basic protein were in- cubated at 37” for 10 hours with trypsin (1% by weight of sub- strate) in 10 ml of ammonium bicarbonate buffer (0.25 M, pH 8.0) containing 0.001 M CaC12. The digestion was terminated by freezing followed by lyophilization.

Peptide Mapping Tryptic Digest-Peptide mapping was per- formed on Whatman No. 3MM paper as described by Katz, Dreyer, and Anfinsen (36). The tryptic digest was subjected to descending chromatography for 26 hours in the short dimen- sion. The upper phase of a n-butyl alcohol-acetic acid-water (4: 1:5, v/v/v) mixture was used as the developing solvent. Electrophoresis was performed in pyridine-acetic acid-water (1: 10: 289)) pH 3.7, at 60 volts per cm for 1 hour. The peptides were visualized by the cadmium-ninhydrin reagent of Atfield and Morris (37) and the arginine-containing peptides were lo- cated with the Sakaguchi reagent (38). A modification of the isatin reagent of Monier and Jutisz was used to determine the number of N-prolyl peptides (39). To 100 ml of a 1% solution of isatin in acetone were added 16 ml of 5 M acetic acid contain- ing 0.05 M cadmium acetate. The peptide map was dipped into the isatin reagent and dried at 60” for 30 min.

Twenty-five milligrams of the complete tryptic digest of the pig brain basic protein were dissolved in 1.0 ml of 0.05 N HCl. Complete acid hydrolysis (24 hours, 6 N HCl, 110”) was per- formed on an aliquot (0.04 ml, 1.0 mg) of the tryptic digest. The quantity of lysine and arginine present in this peptide hy- drolysate was determined on the Beckman-Spinco analyzer ac- cording to the method of Spackman, Stein, and Moore (40). A second aliquot (0.8 ml, 20 mg) of the identical peptide solu- tion was applied directly to a Beckman-Spinco analyzer. The amount of ninhydrin-positive material eluted from the Beck- man-Spinco analyzer at the position of free lysine and free ar- ginine was determined.

To investigate the distribution of proline in the peptides of the pig brain basic protein, descending chromatograms of the tryptic digest were run under the same conditions as those used in the first dimension of the peptide maps. The ninhydrin- positive regions of the chromatograms were identified and ex- cised, and the peptides were eluted by descending chromatog-

raphy with 5% acetic acid. The eluates were evaporated to dryness and hydrolyzed (24 hours, 6 N HCl, 110”) in sealed evacuated tubes (41). The proline content of the hydrolyzed peptides from these regions was examined by paper chromatog- raphy (42).

Preparation of Antisera to Pig Brain Basic Protein-Three female albino rabbits, each weighing 4 pounds, were bled prior to injection to obtain the normal serum used as the control. Each rabbit was injected subcutaneously at seven sites (hind footpads, either side of the lumbar region, above the scapulae, and the cervical fat pads) with 0.2 ml of complete Freund’s adjuvant (43) containing 2 mg of pig brain basic protein (14 mg per rabbit). The same rabbits were then injected intra- venously weekly for 6 weeks with 5 mg of the pig brain basic protein. The rabbits were then bled and the serum was col- lected. The precipitin reaction of these antisera to the pig brain basic protein was investigated by the double diffusion technique of Ouchterlony (43) in 1% ionagar buffered with borate-0.9% NaCl, pH 7.4, and by immunoelectrophoresis (43) in 0.85% ionagar buffered with barbital (pH 8.2, 0.05 M).

Preparation of Fluorescent y-Globulin-Fluorescent y-globulin was prepared according to the method of Riggs et al. (44) from both the immune rabbit serum and from the normal serum ob- tained from the same rabbit prior to injection. The sera were brought to 33% saturation with solid ammonium sulfate at 0”, and the precipitated -y-globulin fraction was collected by cen- trifugation. This precipitate was dissolved to a final concen- tration of 15 mg per ml in sodium bicarbonate, (0.5 M, pH 9.0) and dialyzed against the same bicarbonate buffer for 16 hours.

The dialyzed y-globulin fraction was diluted with an equal volume of 0.9% NaCl, made 10% (v/v) with respect to acetone, and cooled in a Dry Ice-acetone bath until ice crystals began to form. At this point, the acetone concentration was brought to 15% (v/v), fluorescein isothiocyanate was added to a final concentration of 2.5 mg per 100 ml, and the solution was stirred for 24 hours at 4”. The solution was then applied to a column of Sephadex G-25 (1.5 x 30 cm) equilibrated with sodium phos- phate (0.01 M, pH 7.2). The fluorescent y-globulin fraction emerged with the front while the unreacted fluorescein isothio- cyanate remained near the top of the column. The fluorescent y-globulin fraction was mixed with pig kidney acetone powder to remove any nonspecifically reacting material and the resultant suspension was incubated with stirring for 1 hour at room tem- perature. The suspension was cleared by centrifugation, and the fluorescent protein remaining in solution was passed through a 0.35-p Millipore filter to give a clear solution.

Reaction of Fluorescent y-Globulin with Tissue Sections-Fresh samples of the various tissues were rapidly frozen in a cryostat and tissue sections (6 a thick) were cut from the frozen block. The tissue sections were immediately placed in 95 % ethanol at room temperature for 10 min, after which the sections were air-dried. One drop of the fluorescent-immune or fluorescent- normal y-globulin solution was placed on each tissue section, and these sections were incubated in a moist chamber at room temperature for 30 min. An identical procedure was performed with the fluorescent-immune y-globulin after absorption with the pig brain basic protein. The tissue sections were extensively washed in 0.01 M sodium phosphate (pH 7.5) and mounted with glycerol containing 0.001 M sodium phosphate (pH 7.5). The sections were examined by dark field microscopy with an ultra- violet light source having a maximum intensity at 260 mp.

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FIG. 1. Time course of hydrolysis of the pig brain basic protein FIG. 1. Time course of hydrolysis of the pig brain basic protein by trypsin (lo/, by weight of substrate) at 37’. by trypsin (lo/, by weight of substrate) at 37’. The buffer used The buffer used was ammonium bicarbonate (0.25 M, pH 8.0). was ammonium bicarbonate (0.25 M, pH 8.0).

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FIG. 2. Peptide map of the tryptic digest of the pig brain basic protein. The chromatographic direction in the photograph was rotated 180” to facilitate comparison with Fig. 3. Descending chromatography was run for 26 hours with the upper phase of n-butyl alcohol-acetic acid-water (4:1:5, v/v/v) as the devel- oping solvent. Electrophoresis was performed in pyridine- acetate buffer, pH 3.7, at 60 volts per cm for 1 hour. Peptides were visualized with the cadmium-ninhydrin reagent.

FIG. 3. Peptide map of the tryptic digest of the pig brain basic protein. The conditions of chromatography and electrophoresis were identical with those of Fig. 2. Arginine-containing peptides were located with the Sakaguchi reagent.

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FIG. 4. Descending chromatography of the tryptic digests of the pig brain basic protein. Chromatography was run for 26 hours with the upper phase of n-butyl alcohol-acetic acid-water (4: 1:5, v/v/v) as the developing solvent. Peptides were visu- alized with the cadmium-ninhydrin reagent.

I 1 FIG. 5. Tracing of the immunoelectrophoresis of the pig brain

basic protein and its reaction with the specific antiserum. The supporting medium for the electrophoresis was 0.85% ionagar buffered with barbital (pH 8.2, 0.05 M) placed on a microscope slide. One tenth per cent basic protein in the same buffer was placed in the circular wells and subjected to the following elec- trophoretic conditions: 6 volts per cm for 60 min at, 4”. The cathode was to the left and the anode to the right. Antiserum was placed in the trough. The tracing illustrates the single precipitin line that was present. The precipitin line extended from the antigen well toward the cathode.

Photographs were taken with Kodak pan-X plus film. Six- micron sections from the same block were stained with the Paragon multiple stain and examined with the light microscope.

RESULTS

Tryptic Digestion-Digestion of the pig brain basic protein by trypsin was initially very rapid. The ninhydrin-positive ma- terial in samples from the incubation mixture increased from an absorbance of 0.4 at zero time to 0.8 during the first + hour and attained a maximum absorbance of 1.5 at the end of 5 hours of ‘incubation (Fig. 1). All of the peptides produced by tryptic digestion of the pig brain basic protein were soluble under the conditions used in peptide mapping.

Peptide MupsPeptide maps of the hydrolysis products of the pig brain basic protein produced by trypsin are shown in Fig. 2. Under the conditions of peptide mapping described under “Experimental Procedure,” 31 ninhydrin-positive spots (Fig. 2) and 18 Sakaguchi-positive spots (Fig. 3) were visualized. The three ninhydrin-positive regions designated by the arrows (Fig. 2) consist of more than one peptide because only a portion of these regions showed a positive reaction when the peptide maps were stained with the Sakaguchi reagent (Fig. 3). Thus, from peptide maps of tryptic digests, 34 spots may be visualized. The mobility of the ninhydrin-positive material in Spot 1 (Fig. 2) was identical with that of free lysine. The material in Spot d (Fig. 2) migrated as free arginine, and also gave a positive Saka- guchi reaction (Fig. 3). Amino acid analysis was performed on both the tryptic digest and a complete acid hydrolysate of the tryptic digest. On amino acid analysis of the complete tryptic

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2510 Characterization and Immunochemical Localization of Bask Protein from Pig Bruin. II Vol. 243, No. 10

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FIG. 6. Photomicrograph illustrating the appearance of the cell bodies (Ce) of neurons in dorsal root ganglion of a rat. The nucleolus

may be identified as a darkly staining round body within the nucleus @VU). Paragon multiple stain. X 300.

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Issue OP May 25, 1968 L. G. Tomasi and S. E. Korngutfi 2511

digest, symmetrical peaks were obtained at positions correspond- ing to those of free lysine and free arginine. If all of the nin- hydrin-positive material emerging at the lysine and arginine positions represented free lysine and arginine, then 4% of the total lysine and 16% of the total arginine in the tryptic digest existed as the free amino acid.

The neutral amino acid alanine was run under identical condi- tions as were used in peptide mapping. The coordinates of its position on a peptide map are shown at the border of Fig. 2. In comparison to the projected position of alanine on t,he pep- tide map, the position of the peptides in Region A (Fig. 2) in- dicated that the chromatographic mobility of these reptides was lees than alanine whereas the electrophoretic mobility of these peptides was greater than alanine. In addition, the pat- tern of the peptides contained in Region A shows a stacking phenomenon, in that the peptides appear to be positioned in columns in both t,he electrophoretic and chromatographic direc- tions.

Distribution of Proline-The proline content of the pig brain basic protein is 7.8 moles per 100 moles of amino acid (32). Pep- tide maps of the tryptic digest of the pig brain basic protein, sta,ined with the isatin reagent, indicated that none of the tryptic peptides had an imino-terminal proline. The distribution of proline in the tryptic peptides was investigated by analyzing the proline content of the 10 regions resolved by the first dimen- sion of peptide mapping (Fig. 4). Paper chromatograms of the acid-hydrolyzed peptides from these regions stained with the isatin reagent indicated that the proline content of these regions approximated the proline content of the pig brain basic protein.

Immunochemical and Immunohistological Studies-With the double diffusion studies (Ouchterlony met’hod) only one precipi- tin line was observed between the well containing the purified basic protein from pig brain and the well cont,aining the antiserum to this protein. This was the only precipitin line that appeared during the incubation, and the position of this precipitin line with respect to the wells remained constant. No precipitin line was Feen with the normal serum from these Fame rabbits. The immunoelectrophoresis (Fig. 5) showed a single precipitin line extending from the antigen well toward the cathode. The migration of the basic protein at pH 8.2 is at the position at which the precipitin line was observed.

Fluorescent-immune or fluorescent control y-globulin solution was layered over frozen sections of the nervous tissue from monkey, pig, cow, rat, guinea pig, tadpole, and frog. The areas of the nervous system studied in these animals were the cerebrum, cerebellum, brain stem, spinal cord, and dorsal root ganglion. Six-micron sections of the testicle, ovary, liver, spleen, and kidney from the cow and the pig were also studied

by this technique. When control, fluorescein-labeled y-globulin was reacted with sections of the nervous system or the testes, a dull green fluorescence which was of constant intensity was observed in all of the identified morphological structures. This was in contrast to the areas of intense green fluorescence (white areas in micrographs) observed in sections of nervous system and testes that were reacted with the immune fluorescein-labeled y-globulin (Figs. 7 to 13). The intensity of t,he fluorescent light from an area is proportional to the amount of fluorescent-immune y-globulin adsorbed to that area. When fluorescent-immune y-globulin solution was absorbed wit.h the pig brain basic protein and subsequently reacted with the nervous system, a dull green fluorescence was observed. This dull green fluorescence was identical with that obtained after reaction of the nervous system with control fluorescein-labeled y-globulin. The nervous sys- tem in all of the animals investigated gave evidence of a specific reaction with the immune fluorescein-labeled y-globulin. Of the tissues other than those of the nervous system that were examined, only the testes gave evidence of any specific reaction. Sections of ovary, liver, spleen, and kidney gave a dull green fluorescence with both the normal and the immune fluorescent y-globulin.

The neurons of the dorsal root ganglion stained with Paragon multiple stain (Fig. 6) show large nuclei with prominent nucleoli. These neurons are surrounded by the heavily myelinated fiber tracts of the dorsal root. Sections of the dorsal root ganglion reacted with the fluorescein-labeled immune y-globulin showed an intense fluorescence which was concentrated in the nucleus of t.he neurons (Fig. 7). An absence of any reaction with the fluorescent anti-pig brain basic protein antibody in both the Schwann cells and the myelinated fiber tracts of the dorsal root ganglia may also be noted (Fig. 7). Wit,hin t.he central nervous system, fluorescence was observed in the nucleus of neurons from the brain stem (Fig. 8) and the ventral horn of the spinal cord (Fig. 9). A single discrete area of less intense fluorescence was not,ed in many of the nuclei of t)he large ventral horn cells in the spinal cord (Fig. 9). The histological appearance of this area of less intense fluorescence is similar to the nucleolus of the neurons identified by phase microscopy. Less inten:e but posi- tive reactions with the fluorescent anti-basic protein antibody were also noted in both the cytoplasm and axoplasm of these neurons (Figs. 8 and 9). The reaction of the axoplasm with the fluorescent anti-basic protein antibody was the most intense in sections of the dorsal horn of the spinal cord (Fig. IO). In sections of the cerebellum (Fig. II), the entire nuclei of both the large Purkinje cells and the neurons t,hat form the granular layer (granule cells) fluoresced st.rongly. The cerebrum of the frog (Fig. 12) also reacted specifically with the fluorescent-im-

FIGS. 7 TO 13. Photomicrographs of nervous system and testis reacted with the fluorescent anti-pig basic protem anl,ibody. The white regions are the areas which have reacted with the fluorescent-immune r-globulin.

FIG. 7. Photomicrograph of a dorsal root ganglia from a rat. tensely fluorescent nuclei (arrow). X 300.

A loop of fluorescence may be seen extending from one of the in-

FIG. 8. Photomicrograph of a region from the brain stem of a rat. Intense nuclear fluorescence may be seen. cell body appears 8s a halo about t.he intensely fluorescent nucleus.

The cytoplasm of the x 300.

FIG. 9. Photomicrograph of the ventral horn of the spinal cord from a monkey. may be observed in the nucleus of one of the large ventral horn cells.

A circular area of lees intense ffltorescence (arrow) X 500.

FIG. 10. Photomicrograph of the dorsal horn of the spinal cord from a monkey. The fluorescence of the axoplasm (arrow) in this area of the nervous system is equal to the intense fluorescence of the nuclei observed in all areas of the nervous system. x 300.

FIG. 11. Photomicrograph of the cerebellar cortex from a rat. oresce strongly. X200.

The nuclei of the Purkinje (Pu) cells and the granule cells (Gr) flu-

FIG. 12. Photomicrograph of the cerebrum from a frog. The nuclear fluorescence may be seen. X 300. FIG. 13. Photomicrograph of the testis from a pig.

tense fluorescence within the seminiferous tubule. The fluorescence within the nuclei of the spermatogonia srlrround the less in-

X 125.

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mune y-globulin. In the testes (Fig. 13), a distinct fluorescence of this discrepancy to their high content of proline have been was observed in the cells surrounding the seminiferous tubules. discussed. The elution of the lysine-rich histones from Amber- The intensely fluorescent cells are the spermatogonia and the lite IRC-50 at a concentration of 10% guanidinium chloride less intense fluorescence within t,he seminiferous tubule may be (52, 54) is another point in common with the histone isolated due to cells in the more advance stages of spermatogenesis. from pig brain (32). Neither the lysine-rich histones (55) nor

the histones isolated from pig brain gave a “core” of insoluble DISCUSSION peptides upon digestion with trypsin. This is in contrast to all

Peptide maps of the pig brain basic protein after tryptic of the other histone fractions investigated (56). A stacking hydrolysis were used in the further characterization of this phenomenon (peptides positioned in columns in both the elec- protein. The minimum molecular weight of the pig brain basic trophoretic and chromatographic directions) is evident in the protein calculated from its amino acid composition is 13,500 peptide maps of the tryptic digests of the histone isolated from (32) and, at this molecular weight, 1 mole of protein contained pig brain. A similar phenomenon is observed in the same region 12 moles of arginine and 10 moles of lysine. Eighteen arginine- of the peptide maps of the lysine-rich histone Fraction I, follow- containing spots (Sakaguchi-positive) and 16 lysine-containing ing tryptic digestion (53). This phenomenon is not observed spots were identified on peptide maps of the tryptic digests of in the peptide maps of other histone fractions (53). the pig brain basic protein. This is consistent with a protein The fluorescent.-immune y-globulin showed a specific reaction molecule of molecular weight 27,000 (twice the minimum molecu- with the nuclei of the neurons in the nervous systems of the lar weight) which would contain 24 molecules of arginine and 20 monkey, pig, cow, rat, guinea pig, tadpole, and frog. A specific molecules of lysine per protein molecule. At the molecular reaction was also noted in the nuclei of the spermatogonia in the weight of 27,000, amino acid analysis indicated that the maximum test,es of both the pig and the rat. Thus it is concluded that amount of free basic amino acids released from the protein by a protein which is antigenically similar to the histone isolated tryptic digestion was 1 lysine residue (4% of the total lysine) from pig brain is present in the nervous system and in the testes and 4 arginine residues (16% of the total arginine) per molecule of these animals. From the absence of fluorescence in sections of protein. The above results account for 21 of the 24 arginine of the liver, spleen, ovary, and kidney of these same animals, and 16 of the 20 lysine residues available at the postulated it is concluded that a protein which is antigenically similar to molecular weight of 27,000. this histone is either absent or it exists in such a conformation

In a previous study (32), it was reported that the molecular that its antigenic sites are not available to the fluorescent-im- weight of the basic protein, as estimated from ultracentrifugation, mune y-globulin. Lewis (57) showed by complement fixation was 20,000 while the molecular weight of the protein as estimated techniques that antisera against brain extracts reacted with by gel filtration on Sephadex G-100 was 50,000. The apparent testicular extracts and that antisera against testicular extracts high estimate of the molecular weight obtained by gel filtration reacted with brain extract,s. The reaction of the anti-brain has been shown not to be due to aggregation (32). A similar sera and anti-testicular sera to brain or testes is species-inde- discrepancy has occurred in estimating the molecular weight pendent, but neither the anti-brain sera nor the anti-testicular of other highly charged proteins which contain large amounts sera reacted with extracts of the liver, lung, heart, spleen, kid- of proline. Examples of these classes of proteins are the lysine- ney, and ovary (57). These data are consistent with the postu- rich histones from calf thymus (9, 45), the cY-amylase of Bacillus late that the histone isolated from pig brain is a tissue-specific steurothermophilus (46), and a basic protein of the bovine spinal histone. cord (47). Both the lysine-rich histones (48, 49) and the a- The conclusion that histones isolated from different organs are amylase of B. stearothermophilus (46) have been characterized identical has been made solely on the basis of electrophoretic as flexible disordered proteins with a low a-helical content. mobility (15, 16), amino acid composition (17), and amino- These results are consistent with the postulate that the apparent terminal amino acid analysis (17). From this information, it high estimate of the molecular weight of the pig brain basic has been suggested that the biological specificity of histones is protein obtained by gel filtration is due to the nonspherical dependent upon N-methylation (19, 21, 22) or phosphorylation conformation of this molecule. (18, 20, 23) rather than upon the primary sequence of the pro-

The pig brain basic protein was antigenic for rabbits, and the tein. However, the three above techniques by themselves are immune y-globulin produced by these rabbits was coupled with not sufficient to est.ablish whether two protein populations have fluorescein isothiocyanate. With the aid of fluorescent mi- the same primary sequence, although they are useful in clarifying croscopy, it was shown that the fluorescent anti-basic protein possible differences between such populations. Kinkade and antibody was specifically adsorbed to the nuclei of neurons in Cole (54) have recently resolved the lysine-rich histone fraction sections of nervous tissue and to the nuclei of spermatogonia into four distinct fractions with ion exchange chromatography. in Feetions of testes. From the properties of the pig brain basic These four fractions have a similar electrophoretic mobility in protein and its localization within the nucleus of a cell, it is different systems and a similar amino acid composition, although concluded that the pig brain basic protein is a histone (50, 51). these fractions clearly contain different proteins. The present On the basis of its amino acid composition, the pig brain basic report describes the preparation of a specific antibody to a basic protein would be classified as a slightly arginine-rich histone protein which has been shown to be homogeneous by the criteria because the ratio of arginine to lysine in the molecule is 1.2. of ultracentrifugation, starch gel electrophoresis, polyacrylamide However, the properties of the pig brain basic protein show many gel electrophoresis, and end group analysis. This basic protein, similarities to the histones designated as Fraction I, (lysine- which has properties similar to those of the lysine-rich histones, rich histones) by Murray (52, 53). The similar discrepancy has been localized in the nuclei of neurons by immunohistological in estimating the molecular weight of these lysine-rich histones techniques. An antigenetically similar protein was detected and of the histone isolated from pig brain and the relationship in the nuclei of neurons from the nervous system of monkey,

2512 Characterization and Immunochemical Localization of Basic Protein from Pig Brain. II Vol. 243, No. 10

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cow, rat, guinea pig, frog, and tadpole and in spermatogonia from the testes of pig and rat. The protein could not be detected by this technique in the ovary, liver, kidney, and spleen of these same animals. From these data, it is postulated that the basic protein isolated from pig brain is a tissue-specific histone.

~lcknowledgments-We would like to express our appreciation t.o Dr. John W. Anderson and Dr. H. F. Deutsch for the use of equipment during this investigation, to Mr. Richard M. Burton for his critical discussion, and to Miss Judith Oldenburg and Grayson Scott for their valuable assistance. We are also grate- ful to Oscar Mayer and Company (Madison, Wisconsin) for the fresh pig brains.

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Pig Brain: II. PEPTIDE MAPS AND TISSUE-SPECIFIC NUCLEAR Characterization and Immunochemical Localization of a Basic Protein from

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