Histamine-releasing activity. IV. Molecular heterogeneity of the activity from stimulated human thoracic duct lymphocytes

Previous studies with the lymphokine, histamine-releasing activity (HRA), showed that HRA consisted of a heterogeneous group of molecules. The possibility of using thoracic duct lymphocytes (TDL) as a source of large quantities of HRA has been investigated. Antigen-stimulated TDL synthesize and release HRA in quantities similar to an equivalent number of peripheral blood lymphocytes (PBL). Streptokinase (SK) antigen routinely caused TDL to produce HRA approximately 15,000 Da. In contrast, staphylococcus enterotoxin B (SEB) induced the formation of a heterogeneous mixture of HRAs with apparent molecular weights of 50,000 and 15,000. Two peaks of activity (HRA I and II) were recovered when the supernatant from SK-stimulated TDL was subjected to ion-exchange chromatography. Interestingly, basophil chemotactic activity (BCA) was also eluted in these two peaks. Although interferon (IFN) is also released by antigen-stimulated TDL, the nonidentity of IFN and HRA was established by fundamental differences in chromatographic properties and specific antisera to IFN. In contrast, these studies suggest that HRA and BCA may be present on the same molecular entity.     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further characterization of the ataxia-telangiectasia clastogenic factor by reversed-phase liquid chromatography

The clastogenic factor present in medium conditioned by ataxia-telangiectasia (A-T) fibroblast cultures was chromatographed on LiChrosorb RP-8 columns and was eluted with a solution of 20% methanol in 0.005 M NH₄HCO₃. Based on this property, the A-T clastogenic factor was isolated from a C₈ column by high-performance liquid chromatography (HPLC). A specific fraction of the HPLC eluate contained the clastogenic factor. This method makes possible the purification of the A-T clastogenic factor for further analysis.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine triphosphatases associated with bovine brain microtubules. I. Presence of two distinct ATPases and their partial purification

Brain microtubules purified by cycles of assembly and disassembly contained an ATPase activity in the fraction of microtubule-associated proteins (MAPs). This ATPase activity was found to be stimulated by 6S tubulin in the presence of Ca2+ ions, suggesting its functional association with brain microtubules (Ihara et al. (1979) J. Biochem. 86, 587-590). On further purification by DEAE-cellulose column chromatography, two peaks of ATPase activity were separated; one, eluted at 0.2 M KCl (ATPase I), was dependent on added 6S tubulin but the other, eluted at 0.5 M KCl (ATPase II), was not. ATPase I was highly unstable but could be stabilized by the addition of 0.1 mM ADP, 50% (v/v) glycerol or 0.3 mg/ml tubulin. ATPase I was further purified by CM-cellulose column chromatography, and by gel filtration on Sephacryl S-300. Its molecular weight, estimated by gel filtration, was 33,000. ATPase II had a high molecular weight and appeared to be associated with membrane vesicles. It sedimented on glycerol density gradient centrifugation with an s value of 27S. It was purified by high speed sedimentation and hydrophobic chromatography, and was observed under an electron microscope to consist of membrane vesicles of about 70 nm in diameter containing knob-like structures similar to those of H+-pump ATPase.     

Porcine follicular fluid contains several low molecular weight inhibitors of follicle-stimulating hormone binding to receptor

Porcine follicular fluid (PFF) inhibited the binding of 125I-human follicle-stimulating hormone (hFSH) to receptor in vitro in a dose-dependent fashion. PFF (2.5 l) was fractionated on the basis of apparent molecular weight (Mr) by ultrafiltration using hollow fibers and membranes of precalibrated pore size. Desalted, low Mr (500-5000) subfractions containing FSH-binding inhibitor (FSH-BI) activity were further purified by Sephadex G10 gel filtration and anion-exchange high-performance liquid chromatography (HPLC). This resulted in the partial purification of several low Mr FSH-BIs. Three major peaks of FSH-BI were resolved on the Sephadex G10 column eluted with water; G10-1 [elution volume (Ve)/exclusion volume (Vo) = 1.1] had only FSH-BI activity, while G10-2 (Ve/Vo = 1.4) and G10-3 (Ve/Vo = 1.5) had both FSH-BI and luteinizing hormone (LH)-BI activities. A fourth strongly retarded peak (G10-4; Ve/Vo = 2.7) was also obtained. This latter fraction had only FSH-BI activity and represented less than 1% of the FSH-BI activity applied to the column. No separation of these fractions was obtained when the column was eluted with 10 mM ammonium acetate instead of water, suggesting resolution was due to ion-exchange or hydrophobic interactions with the Sephadex. Anion-exchange (Polyanion SI) HPLC of G10-1, G10-2 or G10-3 samples resolved several fractions with FSH-BI activity. A fraction unretained at either pH 5.0 or 7.0 (HPLC-1) was present in all samples. A fraction strongly retained by the column (HPLC-2) and a fraction eluted between 0.13 to 0.24 M acetate (HPLC-3) were present in G10-1 and G10-2 but not in G10-3. HPLC-4, eluted between 0.32 to 0.36 M acetate at pH 5.0, was detected only in G10-3 samples. The most potent low Mr FSH-BI obtained (HPLC-2) inhibited FSH binding by 50% at a dose of 10 micrograms and was enriched approximately 2500-fold relative to whole follicular fluid. These results indicate that PFF contains several low (500-5000) Mr inhibitors of FSH binding to receptor in vitro which differ on the basis of charge, hormone specificity and possibly molecular size and hydrophobicity.     

Purification of the c-fos enhancer-binding protein.

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.     

Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells

A procedure is described for purification of a factor (D-factor)-inducing differentiation of mouse myeloid leukemic cells (M1) into macrophages from serum-free mouse L929 cell-conditioned medium. The procedure included ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-200 and phenyl-Sepharose column chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel-filtration column. The purified factor gave a single band of protein with a molecular weight of 62,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with biological activity. Its half-maximal concentration for inducing differentiation of M1 cells into macrophages was 1.7 X 10(-11) M. Even at 2.6 X 10(-9) M, it did not induce colony formation of normal bone marrow cells, suggesting that it was distinct from the growth factor for normal precursors of macrophages and/or granulocytes.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.     

Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.     

Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms

DEAE-cellulose chromatography, with or without dithiothreitol and over a pH range of 6.0 to 8.5, resolved two phosphodiesterase activities (peaks I and II) from the soluble fraction of pig coronary arteries. The activity of peak I was increased by calmodulin (3-7-fold), whereas that of peak II was not. Chromatography of peak I on Biol-Gel A-0.5 m columns resolved two peaks of phosphodiesterase activity (peaks Ia and Ib). Peak Ia was eluted in the presence or absence of 0.1 M KCl and was relatively insensitive to calmodulin. Peak Ib was eluted only in the presence of KCl and was sensitive to calmodulin. The substrate specificity and kinetic behavior were the same for peaks I, Ia, and Ib. Repeated gel chromatography of either peak Ia or Ib, under appropriate conditions, yielded a mixture of peaks Ia and Ib. Peak Ia appears to be a reversible aggregate of peak Ib. Gel chromatography of peak II resolved only one phosphodiesterase activity, which was eluted without KCl, was highly specific for cyclic AMP, was not sensitive to calmodulin and migrated differently on the gel column than either peak Ia or Ib. Sucrose density gradient centrifugation of the soluble fraction from pig coronary arteries in the presence or absence of dithiothreitol resolved two peaks of phosphodiesterase activity (6.6 S and 3.6 S) which were similar to peaks I and II separated by DEAE-cellulose chromatography with regard to their substrate specificity and their sensitivity to calmodulin. Upon recentrifugation, each of the two peaks of phosphodiesterase activity gave a single peak of activity which migrated with the same S value as did its parent. These results indicate that the two major forms of phosphodiesterase of pig coronary arteries, which are representative of those found in many tissues, are not interconvertible in cell-free systems.     

Purification of brain-type creatine kinase (B-CK) from several tissues of the chicken: B-CK subspecies

A method for the purification of brain-type creatine kinase (B-CK) from several tissues of the chicken, e.g., brain, retina, gizzard and heart was developed involving (1) an affinity chromatography step on Sepharose Blue from which B-CK was specifically eluted by ADP and (2) a subsequent anion exchange chromatography step on a fast protein liquid chromatography Mono-Q column. Two distinct peaks with B-CK activity, both purified to greater than or equal to 99% homogeneity and displaying specific enzyme activities of 300-400 mumol CP/min/mg 1t pH 7.0 and 25 degrees C, were eluted by a salt gradient at a plateau of 150 mmol/l NaCl. The ratio of the two B-CK peaks varied in a tissue-dependent manner, indicating that in chicken the dimerization of native BB-CK from the two major B-CK subunit species is tissue-specific and nonrandom in neural tissues. The fast, efficient and convenient method for the purification of B-CK at small or large scale, operating at yields of 50-70%, makes the purification of this rather labile enzyme from small amounts of tissues possible and greatly facilitates the subsequent characterization of both major and minor dimeric BB-CK subspecies present in these different tissues.     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Purification of the phosphofructokinase regulatory factors

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.     

Ca2+-protease--an enzyme of the metabolism of cytoskeletal proteins in the olfactory membrane of vertebrates

Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.     

Characteristics and behavior during partial purification of estrogen sulfotransferase of guinea pig liver and chorion

Some characteristics of estrogen sulfotransferases from guinea pig liver and chorion were compared. Liver cytosolic activity was stimulated 10-fold by 25 mM monothiolglycerol and 2-fold by 15 mM MgCl2 or CaCl2, similar to that found previously for chorion. Liver and chorion activities were each eluted as a single peak from fast protein liquid chromatography (FPLC) gel filtration columns at apparent molecular weights of 52,300 and 50,000, respectively. Each was eluted during FPLC anion exchange under single, wide peaks with low recoveries. Liver sulfotransferase activity was eluted from Affi-gel Blue columns in the form of several peaks whereas the chorion activity behaved as a single species. The enzymes from both tissues, when partially purified by gel filtration followed by anion exchange, acted upon estrone and estradiol at the 3-position but activity toward dehydroepiandrosterone and testosterone was minimal or undetectable. Affi-gel Blue chromatography followed by FPLC gel filtration resulted in increases in specific activity of 26- and 90-fold for liver and chorion, respectively. Both enzymes were eluted from agarose-hexane-adenosine 3',5'-diphosphate (PAP-agarose) columns as single peaks. Average increases in specific activity for this column step were 40-fold and 96-fold for the entire eluted peaks of liver and chorion enzyme, respectively. Individual fractions from the PAP-agarose column indicated a specific activity increase of as much as 60-fold for liver and 208-fold for chorion. These latter were markedly unstable and it was not possible to obtain further purification by additional steps. Velocity versus substrate concentration curves for the partially purified enzymes showed complex kinetics, particularly with estradiol as substrate.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.