Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

Purification and characterization of two forms of rat interleukin-2

Rat IL-2 produced by spleen cells in culture with concanavalin A was purified using gel filtration, hydrophobic chromatography, and ion-exchange chromatography. At least two forms of rat IL-2 were found to be separable by ion-exchange chromatography. These two forms have been designated form I and form II. Form I of rat IL-2 was purified by a factor of 1297 and found to have a pI of 6.4. Form II was purified by a factor of 669 and found to have a pI between 5.4 and 6.1. Lectin chromatography was used to demonstrate that these two forms most likely differ in the extent of glycosylation. In the presence of tunicamycin the production of form II was significantly reduced. The two forms of rat IL-2 differ in their abilities to promote a mixed-lymphocyte reaction. Their differences in glycosylation may be the reason for these differences in activity.     

Yeast Glycogen Phosphorylase: Characterization of the Dimeric Form and Its Activation

The purification of yeast glycogen phosphorylase [EC 2.4.1.1] was improved by ethanol precipitation and affinity chromatography on a glycogen-Sepharose column. The purified enzyme gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a subunit molecular mass of 100 kDa. Gel electrophoresis also showed that the major activity of native phosphorylase was ascribed to a dimer of 203 kDa, which was agreed with the value obtained by gel filtration on Sephadex G-200. The yeast phosphorylase showed a high affinity for AMP- Sepharose, whereas the enzyme was specifically inhibited by AMP. This inhibition was competitive with respect to the substrate glucose 1-phosphate and gave a Ki value of 9.3 mm. Activation of the crude extract by phosphorylation with an endogenous phosphorylase kinase indicated that the yeast phosphorylase occurred in a mixture of phosphorylated and non-phosphorylated forms.     

Purification and characterization of thymidine kinase from regenerating rat liver

Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70,000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26,000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 microM. ATP acts as a sigmoidal substrate with a 'Km' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.     

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.     

Properties of two molecular forms of beta-glucuronidase from the mollusc Littorina littorea L.

The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.     

Purification and characterization of a lectin from Phaseolus acutifolius var. latifolius

The lectin from Phaseolus acutifolius var. latifolius has been purified to homogeneity by affinity chromatography using stroma-sephadex, gel filtration and electrofocusing. The purified lectin consists of four subunits of 21 Kd molecular mass each one with 7.4% w/w total carbohydrate. Red cells of human and animal species, are agglutinated but are not inhibited by a series of sugars. Mitogenicity and immunosuppressive activities were demonstrated. Agglutination requires magnesium and calcium ions.     

Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

Affinity chromatography and properties of cGMP-dependent protein kinase from prawn tissues

Using affinity chromatography on 8-(2-aminoethyl)-amino-cAMP Sepharose, the cGMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from tissues of the prawn Palaemon adspersus was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The degree of enzyme purification was 11 200, recovery--6.5%; the isoelectric point for the enzyme lies at 5.5. Data from gel filtration and centrifugation in sucrose density gradient suggest that the dimer of cGMP-dependent protein kinase has a molecular weight of 157 000, sedimentation coefficient of 7.2S and a Stokes' radius of 50 A. An active form of the enzyme with Mr = 76 500 (4.5S, 39 A) which apparently represents a subunit of the cGMP-dependent protein kinase was discovered. The activity of the both enzyme forms are stimulated by low concentrations of cGMP (Ka = 1.10(-7) M). The monomer and dimer molecules appear as prolate ellipsoids with axial ratios close to 7. The native cGMP-dependent protein kinase is probably made up of two subunits each of which contains a regulatory and a catalytic sites.     

Purification and characterization of a high molecular weight type 1 phosphoprotein phosphatase from the human erythrocyte

The major Mn2+-activated phosphoprotein phosphatase of the human erythrocyte has been purified to homogeneity from the cell hemolysate. It is sensitive to both inhibitors 1 and 2 of rabbit skeletal muscle, preferentially dephosphorylates the beta subunit of the phosphorylase kinase, and dephosphorylates a broad range of substrates including phosphorylase a, p-nitro-phenyl phosphate, phosphocasein, the regulatory subunit of cyclic AMP-dependent protein kinase, and both spectrin (Km = 10 microM) and pyruvate kinase (Km = 18 microM) purified from the human erythrocyte. The purified enzyme is stimulated by Mn2+ and to a lesser extent by higher concentrations of Mg2+. The purification procedure was selected to avoid any change in molecular weight, hence subunit composition, between the crude and purified enzyme. Maintenance of the original structure is demonstrated by non-denaturing gel electrophoresis and gel filtration chromatography. Gel filtration of the purified holoenzyme shows a single active component with a Stokes radius of 58 A at a molecular weight position of 180,000. Sedimentation velocity in a glycerol gradient gives a value of 6.1 for S20, w. Together these data indicate a molecular weight of about 135,000. Two bands of equal intensity appear on sodium dodecyl sulfate-gel electrophoresis at molecular weights of 61,700 and 36,300, suggesting a subunit composition of two 36,000 and one 62,000 subunits. The 36-kDa catalytic subunit can be isolated by freezing and thawing the holoenzyme or by hydrophobic chromatography of the holoenzyme. The catalytic subunit shows unchanged substrate and inhibitor specificity but altered metal ion activation.     

A rapid purification method for human erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

Purification and characterization of a type II casein kinase from Drosophila melanogaster

A cyclic nucleotide-independent protein kinase has been isolated from Drosophila melanogaster by chromatography on phosphocellulose and hydroxylapatite followed by gel filtration and glycerol gradient sedimentation. As determined by sodium dodecyl sulfate gel electrophoresis, the purified enzyme is greater than 95% homogeneous and is composed of two distinct subunits, alpha and beta, having Mr = 36,700 and 28,200, respectively. The native form of the enzyme is an alpha 2 beta 2 tetramer having a Stokes radius of 48 A, a sedimentation coefficient of 6.4 S, and Mr approximately 130,000. The purified kinase undergoes an autocatalytic reaction resulting in the specific phosphorylation of the beta subunit, exhibits a low apparent Km for both ATP and GTP as nucleoside triphosphate donor (17 and 66 microM, respectively), phosphorylates both casein and phosvitin but neither histones nor protamine, modifies both serine and threonine residues in casein, and is strongly inhibited by heparin (I50 = 21 ng/ml). These properties are remarkably similar to those of casein kinase II, an enzyme previously described in several mammalian and avian species. The strong similarities among the insect, avian, and mammalian enzymes suggest that casein kinase II has been highly conserved during evolution.     

Isolation and comparative studies of glycogen phosphorylase isoenzymes from lymphocytes and polymorphonuclear leucocytes

Two glycogen phosphorylase isoenzymes have been identified in pig lymphocytes and polymorphonuclear leucocytes by DEAE cellulose chromatography. Both isophosphorylases have been further purified by affinity chromatography on Sepharose-AMP to almost homogeneity. The purified isophosphorylases were composed of subunits of molecular weight similar to the muscle and liver monomers. Isophosphorylase I was more related to the liver enzyme than isophosphorylase II based on immuno-inhibition experiments. Both isoenzymes were markedly different from liver and muscle phosphorylases in their activation by AMP, sodium sulfate and 1,2-dimethoxyethane.     

Purification and characterization of protein phosphatase 1I activating kinase from bovine brain cytosolic and particulate fractions

The activating kinase of protein phosphatase 1I is distributed in approximately equal amounts between the cytosolic and particulate fractions of bovine brain homogenates. Both species of this protein kinase have been purified to near homogeneity. The cytosolic form, purified about 7,000-fold, has an apparent Mr = approximately 75,000, as estimated by gel filtration chromatography on Sephacryl S-300. The enzyme contains two subunits, with apparent Mr = 52,000 and 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both subunits undergo phosphorylation when the enzyme is incubated with Mg2+ and [gamma-32P]ATP. Peptide maps of the two subunits are different, and rabbit antibodies to the 52-kDa subunit show only very minor cross-reactivity to the 46-kDa subunit. These observations indicate that the two subunits are different. The species of protein phosphatase 1I activating kinase that is associated with the membrane fraction has an apparent Mr = approximately 105,000 as estimated by gel filtration. This species also contains two subunits, with apparent Mr = 52,000 and 46,000, the properties of which are very similar, if not identical, to those of the two subunits comprising the cytosolic form of the protein kinase.     

Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species

High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5′ diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I₅₀ = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I₅₀ = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I₅₀ = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg²⁺ and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour.     

Separation and properties of three forms of cathepsin H-like cysteine proteinase from rat spleen

Three forms of cathepsin H-like cysteine proteinase were purified from rat spleen by a method involving acid treatment and chromatography on pepstatin-Sepharose, Sephadex G-75, DEAE-Sephacel, CM-Toyopearl, and concanavalin A-Sepharose. The final preparations of these forms all migrated as single protein bands on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS). The molecular weights of the three forms were estimated to be 28,000 (form I), 26,000 (form II), and 22,000 (form III). The optimal pH was 6.5 for forms I and III and was 7.0 for form II with L-leucine 2-naphthylamide (Leu-NA) or with alpha-N-benzoyl-DL-arginine 2-naphthylamide (BANA). All of the forms consisted of two major species having isoelectric points of 7.1 and 6.5 on isoelectric focusing gels. They were all stable when incubated at pH values between 5.0 and 9.0 for 1 h at 22 degrees C. They were strongly inhibited by iodoacetic acid and E-64, but not by metal ions or pepstatin. Form III was not affected by leupeptin, chymostatin, antipain or elastatinal, which gave essentially complete inhibition of cathepsin B purified from rat spleen. Forms I and II were slightly inhibited by these compounds at the same concentrations. The properties of these forms were compared with those of the known enzymes cathepsin H and BANA-hydrolase.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

A newly synthesized selective casein kinase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification of casein kinase I from bovine testis

When screening various isoquinolinesulfonamide compounds which we synthesized, CKI-7, N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, was found to have a potent inhibitory action against casein kinase I and a much weaker effect on casein kinase II and other protein kinases. Kinetic analysis indicated that CKI-7 inhibited casein kinase I competitively with respect to ATP and that the Ki values were 8.5 microM for casein kinase I and 70 microM for casein kinase II. An affinity chromatography absorbent was synthesized by coupling CKI-8 (1-(5-chloroisoquinoline-8-sulfonyl], a derivative of CKI-7, to cyanogen bromide-activated Sepharose 4B. Partially purified casein kinase I from bovine testis was subjected to affinity chromatography. Analysis of the purified casein kinase I by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single band with molecular weight 37,000. These newly synthesized compounds, CKI-7 and CKI-8, should serve as useful tools for elucidating the biological significance of casein kinase I-mediated reactions.