Early Stages Of Purification Research Articles

Cleavage of proglucagon by the prohormone converting enzymes PCSK1 and PCSK2 is not dictated by the amino acid sequence immediately adjacent to the cleavage site

The peptide hormones glucagon, glucagon‐like peptide‐1, and glucagon‐like peptide‐2 are all derived from proglucagon (PG), a 160 amino acid polypeptide containing multiple potential cleavage sites designated by either single or paired basic amino acids. Initial cleavage of PG takes place on the C‐terminal side of the K70‐R71 dibasic site, which liberates the intermediates glicentin and major proglucagon fragment (MPGF). My laboratory is interested in examining the specificity of the prohormone cleaving enzymes PCSK1 and PCSK2 using both PG and proinsulin (PI) as model substrates. PG is a useful model because it has one cleavage site (the glicentin/MPGF junction) recognized almost equally well by both enzymes and multiple potential cleavage sites that are not recognized by either enzyme. PI is a good model because it has one cleavage site (the B chain/C peptide junction) preferentially recognized by PCSK1 and a second cleavage site (the C peptide/A chain junction) preferentially recognized by PCSK2. My lab has previously described the expression and purification of these two model substrates. However, the purification of PG has presented a number of problems because of the presence of contaminants that obscure detection and participate in co‐precipitation during early stages of purification. I have now devised an improved, two‐step scheme using step elution from SP Sepharose as a capture step, followed by gradient elution from Q Sepharose as the major purification step. This method resolves the problems posed by the contaminants and generates PG samples suitable for analysis of the PCSK‐mediated cleavage reaction. An in vitro cleavage assay was then set up so that the PG substrate (~3 μM) incubated with either PCSK1 or PCSK2 (~100 nM at pH 6.0 or 5.4, respectively) results in ~30% cleavage at the glicentin/MPGF junction using either enzyme. As noted above, in contrast to the situation with PG, the normal maturation pathway for PI exhibits a marked preference for PCSK1‐mediated cleavage at the B chain/C peptide junction. To examine whether this cleavage selectivity is due to amino acids in the immediate vicinity of the cleavage site, the sequence EAED from the C‐terminal side of the B chain/C peptide cleavage site in PI was cloned into the C‐terminal side of the K70‐R71 cleavage site in PG. In contrast to expectations, this substitution resulted in a nearly 50% decrease in PG cleavage by PCSK1. Equally surprising was that this substitution had almost no effect on the efficiency of PCSK2‐mediated cleavage of the altered site, despite the fact that in the original context of the PI substrate, this sequence is recognized much less efficiently by PCSK2 than by PCSK1. Taken together, these results suggest that three‐dimensional features (secondary or tertiary structure) play a significant role in the cleavage site specificity exhibited by both PCSK1 and PCSK2.

Characteristics of starch obtained at different stages of purification during commercial wet milling of maize

Characteristics of starch obtained at different stages of purification during commercial wet milling of maize were evaluated. L*, whiteness index, swelling power, transmittance and syneresis progressively increased while a*, b*, protein content, lipid content and water binding capacity of the starches decreased after each successive purification stage. Protein and lipids content decreased from 5.2 to 0.3% and from 0.15 to 0.03%, respectively, after each successive purification stage. SDS–PAGE indicated that high MW polypeptides eliminated during early stages of purification than low MW. The starches showed typical A-type XRD pattern along with increasing crystalline region after each successive purification stages. DSC evaluation showed the presence of two endotherms during heating, the first between 69.9 and 81.5°C and second endotherm between 84.1 and 105.3°C being associated with the melting of crystallites and amylose–lipid complexes, respectively. Transition temperatures decreased while gelatinization enthalpy increased with the purification of starch. During cooling, exotherms at peak temperature between 78.8 and 90.3°C was observed. Peak viscosity, final viscosity, breakdown viscosity and setback increased while pasting temperature decreased after each successive purification stage. Storage modulus, syneresis and transmittance of starches revealed increased tendency towards retrogradation with decrease in protein content and lipid content.

Mass spectrometry in microbial metabolomic analysis as an analytical tool for dereplication strategy

Metabolomics is the quantitative analysis of wide arrays of metabolites, including seoncdary metabolites, in biological samples. Metabolic profiling focuses on a group of metabolites while metabolic fingerprinting deal with global screening approach to classify samples based on metabolite patterns. The application of MS for analysis of secondary metabolites has grown over the last two decades, and today MS is the most important detector method in biotechnology [1]. Dereplication accelerates the discovery of novel natural products by eliminating repetitive work on known compounds. MS has been used for the dereplication of natural products because it is sensitive and provides information about the molecular mass, molecular formula and substructure of molecules [2], allowing dereplication of natural products at their early stages of purification and characterization. The aim of this study was to analyze metabolite profiles of crude extracts obtained from actinomycetes in differents cultivation medium by direct infusion MS and LC/MS. There are differences in the production of secondary metabolites in response to the growth conditions. The dereplication strategy for the analysis of the secondary metabolites has been developed. First, full scan total ion chromatogram (TIC), scanning m/z 100–1200, was acquired in both positive and negative modes. Ionization of compounds was better in positive mode. The dereplication was carried out in the mass range of m/z 750–900. The data suggest that the secondary metabolites located in this mass range belong to class of macrotetrolides and macrolides antibiotics (Figure 1). Compounds were isolated by a bioassay-guided fractionation using the microalga Chlorella vulgaris as test organism.

Application of RNase in the purification of RNA-binding proteins

Application of RNase in the purification of RNA-binding proteins

Molecular Formula Analysis by an MS/MS/MS Technique To Expedite Dereplication of Natural Products

A facile and sensitive mass spectrometric method has been developed for the dereplication of natural products. The method provides information about the molecular formula and substructure of a precursor molecule and its fragments, which are invaluable aids in dereplication of natural products at their early stages of purification and characterization. Collision-induced MS/MS technique is used to fragment a precursor ion into several product ions, and individual product ions are selected and subjected to collision-induced MS/MS/MS analysis. This method enables the identification of the fragmentation pathway of a precursor molecule from its first-generation fragments (MS/MS), through to the nth generation product ions (MSn). It also allows for the identification of the corresponding neutral products released (neutral losses). Elements used in the molecular formula analysis include C, H, N, O, and S, as most natural products are constituted by these five elements. High-resolution mass separation and accurate mass measurements afforded the unique identification of molecular formula of small neutral products. Through sequential add-up of the molecular formulas of the small neutral products, the molecular formula of the precursor ion and its productions were uniquely determined. The molecular formula of the precursor molecule was then reversely used to identify or confirm the molecular formula of the neutral products and that of the productions. The molecular formula of the neutral fragments allowed for the identification of substructures, leading to a rapid and efficient characterization of precursor natural product. The method was applied to paclitaxel (C47H51NO14; 853 amu) to identify its molecular formula and its substructures, and to characterize its potential fragmentation pathways. The method was further validated by correctly identifying the molecular formula of minocycline (C23H27N3O7; 457 amu) and piperacillin (C23H27N5O7S; 517 amu).

Importance of sialic acid in recombinant thrombomodulin in terms of pharmacokinetics and separation of desialyzed glycoprotein.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.

Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri.

NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of higher specific activity then previously reported. NAH is most stable at alkaline pH in the presence of glycerol. The protein which consists of four dissimilar subunits occurs in forms of different molecular masses. There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein promoter. Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium. Mo(V), flavin radical, and two Fe2S2 clusters could be observed with EPR spectroscopy. The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released from NAH as a reactive low molecular weight compound by a number of denaturing procedures. Parallel losses of Se and catalytic activity were observed during purification and storage of the enzyme. Addition of sodium selenide or selenophosphate did not restore the catalytic activity of the enzyme. Instead, NAH is reversibly inactivated by these compounds and also by sulfide. Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity. The "as isolated" enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detected at early stages of purification. NAH exhibits a high substrate specificity toward electron donor substrates. The ability of a nicotinate analog to reduce NAH (disappearance of 2.067 signal) correlates with the rate of oxidation of the analog in the standard assay mixture. The properties of NAH differentiate the enzyme from known Mo-containing hydroxylases.

Purification, properties, and phosphorylation by protein kinase C of two phosphoinositidase C isozymes from rat brain.

Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5 and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.

3] 3-Hydroxy-3-methylglutaryl-CoA synthase from chicken liver

Publisher Summary This chapter focuses on 3-Hydroxy-3-methylglutaryl-CoA Synthase from Chicken Liver. 3-Hydroxy-3-methylglutaryl-CoA is formed in liver as a result of the essentially irreversible reaction of acetyl-CoA with acetoacetyl-CoA, 1 illustrated in the above equation. The enzyme that catalyzes the reaction, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, reacts reversibly with acetyl-CoA to form an acetyl-enzyme. This species subsequently condenses with the second substrate to form an intermediate which contains HMG-CoA covalently linked to an active site cysteinyl -SH. Hydrolysis of the thioester linkage between enzyme and the condensation product results in release of HMG-CoA and accounts for the irreversible nature of the overall reaction. Modifications that include stabilization of enzyme in the early stages of purification and elimination of ammonium sulfate precipitation as a method of protein concentration have increased the reproducibility and yield of the preparation. An alternative radiochemical assay relies upon the formation of a product ([14C]HMG-CoA), which produces nonvolatile 14 C radioactivity under conditions used to volatilize the unreacted [ 14 C]acetyl-CoA substrate.

Molecular weights of glycollate oxidase from C 3 and C 4 plants determined during early stages of purification

The M r s of glycollate oxidase (EC 1.1.3.1) (GAO) determined soon after extraction from the leaves of several C 3 and C 4 plants are reported. The enzyme isolated from the C 3 plants wheat, barley, spinach, pea and tobacco has M r in the range 160–180 000 and is probably a homotetramer. GAO purified from pea was previously reported as a dimer and as an octamer from spinach leaves. Therefore the quaternary structure of these GAOs soon after extraction differs from that of the purified proteins. The enzymes from the C 4 plants maize and sugar cane have M r s ca twice this value in the range 290–310 000, whilst that of the C 4 grass Panicum maximum has an M r of 162 000. An improved spectrophotometric assay for GAO, using a non-carcinogenic dye, is described.

A basal unit of valine-sensitive acetolactate synthase of [formula omitted

Valine-sensitivity as well as activity of acetolactate synthase of Neurospora crassa was stabilized with 1.2 M potassium phosphate buffer during extraction from mitochondria and early stages of purification, and with 20 % glycerol plus 5 mM sodium pyruvate during Sephadex G200 gel chromatography. The enzyme was expressed as four molecular species having the molecular weights of about 500,000, 140,000, 68,000 and 51,000, respectively. The first and the third species showed valine-sensitivity, but the second and the fourth did not. The third molecular species with a molecular weight of 68,000 may be the basal unit of valine-sensitive acetolactate synthase of Neurospora crassa .

Studies on the Purification of Chitin Synthase from Coprinus cinereus

Chitin synthase has been characterized from the stipes of Coprinus cinereus by a number of techniques. In the absence of digitonin, on gel filtration columns and during electrophoresis the enzyme showed properties consistent with having molecular weights from 1.5 105 to several million, suggesting its reversible aggregation into large multimolecular units. Gel chromatography in buffers containing digitonin gave highly reproducible results, and when followed by anion-exchange chromatography gave preparations with very high activity [e.g. 3.4 mol substrate incorporated min-1 (mg protein)-1] and apparent molecular weight 8.0 105. The best purification (140-fold) was achieved by gel chromatography followed by copper chelate affinity chromatography, giving a nearly pure enzyme preparation of activity 4.7 mol substrate incorporated min-1 (mg protein)-1, which showed only one band of molecular weight 6.7 104 on SDS-polyacrylamide electrophoresis. These purified preparations were free of the nucleoside diphosphatase and protease activities that were present in the early stages of purification.

Phosphorylation and immunology of poly(A) polymerase

Phosphorylation of poly(A) polymerase by protein kinase NI (a cyclic nucleotide-independent nuclear kinase closely associated with poly(A) polymerase at early stages of purification) resulted in as much as 7-fold activation of poly(A) polymerase. Phosphorylation causes an increase in the rate rather than the extent of polyadenylation. Antibodies raised in rabbits against purified poly(A) polymerase from Morris hepatoma 3924A reacted specifically with poly(A) polymerase following “Western” transfer of the enzyme onto diazobenzyloxyl methyl paper. Using iodinated enzyme, a competition radioimmunoassay for poly(A) polymerase was developed. Using the radioimmunoassay, it was shown that Morris hepatoma 3924A contains 100 μg of poly(A) polymerase/mg DNA or 10 7 molecules of the enzyme/cell nucleus. Nuclear poly(A) polymerase from fetal liver, but not from normal liver, was able to compete well with hepatoma enzyme in the radioimmunoassay. These data suggest that the tumor poly(A) polymerase is probably an oncofetal antigen, resulting from derepression of a gene not normally expressed in adult liver.

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468–7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833–2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of M r 42000 and 25000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.

Stimulating factor for calf thymus DNA polymerase β

A factor that stimulates purified DNA polymerase β about 2-fold was separated from DNA polymerase β activity on a DNA-cellulose column. During the early stage of purification, the factor may be associated with DNA polymerase β to form a complex that sediments at 3.9 S in sucrose gradients and behaved as a 52,000 dalton protein on a Sephadex G-100 column. The complex, which contains 40,000 and 12,500 dalton polypeptides, was insensible to the stimulator, and did not show any exonuclease activity.

Sorbin, a peptide contained in porcine upper small intestine which induces the absorption of water and sodium in the rat duodenum.

A fraction increasing water and sodium absorption in rat duodenum was detected in the material obtained at an early stage of purification of the hitherto isolated duodenal hormones. In Wistar rats, duodenal loops were made in situ and filled with a solution containing 0.138 mM NaCl, with 14C PEG and 22Na as markers; the final content was collected after 1 h and the movements of water and Na measured. In contrast to secretin, cholecystokinin, and somatostatin, which induced duodenal secretion, and with pentagastrin, which induced duodenal absorption and stimulated acid secretion, this fraction induced duodenal absorption f Na and water without stimulating acid secretion. The fraction was obtained by chromatography of a concentrate of intestinal peptides in 0.2 M acetic acid on Sephadex G25 (fine), and its active component was found to be methanol-soluble at pH4 and insoluble at pH7.5. It was eluted from carboxymethylcellulose 22 with 0.04 M ammonium bicarbonate and gel filtration of Sephadex G50 *fine), resulting in a tenfold increase in activity. Incubation with chymotrypsin suppressed the biological activity, indicating a peptidic nature. The substance displayed biological and radioimmunological properties distinct from those of the gastrointestinal hormones. Particularly, no cross-reactivity was found with gastrin, prolactin, and angiotensin, which are known to increase intestinal absorption. It therefore seems possible that the activity described is due to a peptide that has as yet not been isolated. The name 'sorbin' is proposed for this active principle.

Phosphoprotein Phosphatase of Soybean Hypocotyls

A soybean histone-type protein kinase was used to prepare (32)P-labeled histone H1 as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.1.3.16) from soybean hypocotyls. The phosphatase has been purified 169-fold by ammonium sulfate fractionation, ethanol precipitation, and chromatography on Sephadex G-150, DEAE-Sephadex A-25 and Sephadex G-100. The activity of the phosphoprotein phosphatase is distinct from that of acid and alkaline phosphatases (EC 3.1.3.1) as well as from that of nucleotidases. The final enzyme preparation does not contain histone protease activity, although it can be detected during the early stages of purification. The protease(s) apparently can attack phosphorylated histone H1, indicating that phosphorylation does not protect the protein against proteolytic degradation.The amounts of (32)P released from [(32)P]histone H1 are proportionally recovered as [(32)P]Pi, indicating that the dephosphorylation is due to the action of phosphoprotein phosphatase. The enzyme shows maximal activity at pH 7 to 8 and has a specific activity of 19 nanomoles of [(32)P]Pi released from [(32)P]histone H1 per minute per milligram of protein. The apparent K(m) for phosphohistone H1 is 4.0 +/- 0.4 micromolar. The estimated molecular weight of the enzyme is approximately 30,000 by gel filtration. The enzyme activity does not depend upon the addition of reducing agent and metal ion. Zn(2+), Co(2+), NaF, pyrophosphate, or ATP at 1 millimolar, however, inhibits the enzyme activity by about 70%. The enzyme activity is unaffected by cyclic nucleotides and plant growth substances but is inhibited by polyamines. All the phosphorylated histone species and protamine, not low molecular weight phosphoesters, act as competitive inhibitors for the dephosphorylation of [(32)P]histone H1.Besides its action on phosphohistone H1, the soybean enzyme also catalyzes the dephosphorylation of other phosphohistone species (H2A, H2B, H3, and H4), degraded phosphohistone H1, and possibly phosphorylated casein and phosvitin. All these results indicate that the enzyme is a nonspecific phosphoprotein phosphatase.

46] Synthesis and isolation of 8α-substituted flavins and flavin peptides

Publisher Summary This chapter provides information on the synthesis and isolation of 8α-substituted flavins and flavin peptides. The presence of covalently bound flavin in succinate dehydrogenase was demonstrated in 1955 but a complete proof that its structure is 8α-[N (3)-histidyl]-FAD was published after some 15 years. Determination of flavin fluorescence has manifold use in the field of 8α-substituted flavins. First, it may provide a convenient means of estimating the quantity of flavin peptide during the early stages of purification in the presence of contaminating materials, such as heme peptides, which interfere with spectrophotometric determination. Second, it is often possible to predict whether a histidyl or cysteinyl residue is involved in the linkage. Despite these advances, the determination of the structure of a new type of covalently bound flavin remains a difficult task, particularly in enzymes containing a cysteinyl flavin, because these tend to be very unstable, particularly after hydrolysis to the aminoacyl flavin level.

Separation and Characterization of Two Phosphorylase Phosphatase Inhibitors from Rabbit Skeletal Muscle

Two heat-stable and trypsin-labile inhibitors of phosphorylase phosphatase, designated inhibitor-1 and inhibitor-2, were partially purified from extracts of rabbit skeletal muscle by heating and coloumn chromatography using DEAE-dellulose and Bio-gel P-60. Inhibitor-1 exists in an active phosphorylated form and an inactive dephosphorylated form. The interconversion of phosphorylated inhibitor-1 and dephosphorylated inhibitor-1 is mediated by protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and a Mn2+-stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor-2 is not influenced by treatment with either the kinase or the Mn2+-stimulated phosphatase. The molecular weights of inhibitor-1 and inhibitor-2 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor-1 and inhibitor-2 inhibit phosphorylase phosphatase by a mechanism which appears to be non-competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic-AMP-dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor-1 and inhibitor-2.