Purification and properties of pyruvate kinase type M2 from rat lung

(1) Pyruvate kinase type M₂ from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M_r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M₂, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K_0.5 value for phosphoenolpyruvate is 0.26 mM (n_H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (n_H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The K_m value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The K_m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M₂ from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M₂ from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M₂ may represent a separate form of the pyruvate kinase type M₂ family.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

Summary A method for the localization of pyruvate kinase isoenzymes type L, M₂ and M₁ in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M₁ was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M₁. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M₂ was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M₂ was detected in high concentrations in the granulation tissue of type M₂ was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M₂ in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Pancreatic islets contain the M2 isoenzyme of pyruvate kinase its phosphorylation has no effect on enzyme activity

To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M₂ isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M₁) and liver (type L). The islet pyruvate kinase was inhibited by anti-M₁ pyruvate kinase serum (which crossreacts with the M₂ isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M₂ isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M₂ and M, isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.Abbreviations EGTA ethylene glycos his (-aminoethyl ether)-N,N,NN-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Pyruvate kinase isozymes in man

Summary By focusing in sucrose, gradient L-type pyruvate kinase from human liver could be separated into 2 major forms (pI 6.28±0.03 and 5.85±0.09) and a minor more acid form (pI5). These different forms could also be detected by focusing in acrylamide-ampholine slab gel. The major forms were interconvertible, the equilibrium being shifted toward the acid form by fructose 1,6-diphosphate and SH reagents, and toward the alkaline form by proteinic factors extracted by ammonium sulphate fractionation from liver extracts and from hemolysates. These factors seemed to be responsible for the stabilization of the liver crude extract enzyme in its alkaline conformation.By acrylamide slab gel electrofocusing, erythrocyte pyruvate kinase from whole hemolysates exhibited a complex pattern composed of at least 3 interconvertible forms. The in vitro aging of the red blood cells and the storage of the hemolysates resulted in a progressive disappearance of the acid forms and in a strengthening of the alkaline form. Partially purified erythrocyte enzyme focused in 2 major bands, interconvertible under the influence of the same factors as those described for L-type pyruvate kinase. Although closely related, the focusing patterns of L-type and erythrocyte-type were never exactly identical.Double immunodiffusion against antihuman L-type serum showed a complete identity reaction between erythrocyte-and L-type pyruvate kinases. Moreover, antihuman M₂-type serum was unable to neutralize erythrocyte pyruvate kinase as well as to change its electrophoretic mobility.Consequently, we conclude that both human erythrocyte-and liver L-type pyruvate kinases existed under several conformers interconvertible under the influence of the same ligands or proteinic factors; erythrocyte-type enzyme seems to include L-type subunit and not M₁- or M₂-type subunits. The erythrocyte- and L-type enzymes, however, are not identical and the nature of the differences between them is discussed.Chargé de recherche INSERM.     

Pressure-adaptive differences in the binding and catalytic properties of muscle-type (M4) lactate dehydrogenases of shallow- and deep-living marine fishes

Summary The pressure sensitivities of substrate (pyruvate) and cofactor (NADH) binding and catalytic rate of purified muscle-type (M₄) lactate dehydrogenases (LDH, EC 1.1.1.27; NAD⁺: lactate oxidoreductase) from shallow- and deep-living teleost fishes were compared. The LDH's of the shallow species are significantly more pressure-sensitive than the LDH's of the deep-living fishes. The apparent Michaelis constant (K _m)¹ of pyruvate of the deep-living species' LDH's is pressure-insensitive over the entire pressure range used in these studies, 1 to 476 atmospheres (Fig. 1). For the LDH's of the shallow species, theK _m of pyruvate increases significantly between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres. TheK _m of NADH displays a much higher pressure sensitivity. For the LDH's of the deep species, theK _m of NADH increases slightly (approximately 32%) between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres (Fig. 1). TheK _m of the shallow species' LDH's rises sharply (approximately 113%) between 1 and 68 atmospheres, and then continues to increase at a slower rate up to 476 atmospheres. This marked inhibition of cofactor binding by pressure for the shallow species' LDH's may be of sufficient magnitude to seriously impair the function of these LDH's at pressures typical of those encountered by the deeper-living species.Pressure effects on optimal velocity, measured under high (optimal) concentrations of pyruvate and NADH, were generally lower for the LDH's of the deep species (Table 1).These results indicate that M₄-LDH's of shallow water fishes are not pre-adapted for function at deepsea pressures, and that the reduction of pressure sensitivities ofK _m's and catalysis may be a ubiquitous feature of adaptation to life at depth. The virtually identical pressure responses of M₄-LDH's from deepliving teleosts belonging to four different families represents a striking example of convergent evolution at the molecular level.     

Bovine pyruvate kinase isozymes and hybrid isozymes. Electrophoretic studies and tissue distribution.

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K₄, L₄, and M₄), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K₂L₂ and K₃M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K₄ also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M₄. The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M₄ with a little KM₃. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K₃M, while Purkinje fibers have a five-membered set dominated by KM₃. The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.     

Ribonucleoprotein complexes of pyruvate kinase from human skeletal muscle

Summary About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase M_B and water soluble form M_A.Crystalline nucleoproteins are composed of active and inactive RNA-protein complexes with varying RNA content. By fractional crystallization and gel filtration a number of crystalline complexes were separated, two of them highly purified. One preparation was homogenous, contained 0.5% RNA and had a specific activity of 265 U/mg protein, the other one 10% RNA and 200 U/mg protein respectively.Forms M_A and M_B share the same protein as shown in immunodiffusion test with the anti-M_A sera. They differ in solubility and stability in diluted solutions. In Tris-HCl buffer, pH 7.6 form M_B is rapidly inactivated whereas form M_A is quite stable under the same conditions. Both forms have different K_m for phosphoenolpyruvate and ADP and V_max as well.Digestion of pyruvate kinase M_B with RNase was without marked effect on specific activity of the enzyme.The presence of numerous ribonucleoprotein complexes with a polynucleotide content in the range of 0.5 to 20% and specific activity of 160–220 U/mg protein suggests the control by RNA binding of pyruvate kinase activity from human skeletal muscle.This work was supported by a grant from the Biochemical and Biophysical Committee of the Polish Academy of Sciences.     

Differences and similarities in binding of pyruvate and l-lactate in the active site of M4 and H4 isoforms of human lactate dehydrogenase

We present QM/MM calculations that show differences in geometries of active sites of M₄ and H₄ isoforms of human LDH ligated with oxamate, pyruvate or l-lactate. As the consequence of these differences, binding isotope effects of the methyl hydrogen atoms of pyruvate and l-lactate may be used to experimentally distinguish these isoforms. Based on the FEP calculations we argue that l-lactate is a better candidate for the experimental studies. Our calculations of energies of interactions of ligands with the active site residues provide explanation for the observed experimentally sensitivity to inhibition of the M₄ isoenzyme isoform and pinpoint the differences to interactions of the ligand with the histidine residue. We conclude that pyruvate interacts much stronger in the active site of H₄ than M₄ isoform and that the latter interactions are weaker than with water molecules in the aqueous solution.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M₁-, M₃-, and M₅-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70 ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M₁- and M₅-acetylcholine receptors and the amplitude of these signals was larger at the M₁-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M₁-, M₃- and M₅-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.     

The control by RNA of pyruvate kinase activity in human skeletal muscle

Summary About 25% of total pyruvate kinase activity in human skeletal muscle is associated with the ribonucleoprotein complexes soluble in salt solutions of high ionic strength. These complexes, called form M_B, crystallize readily from 48% saturated ammonium sulfate at pH 5.6.Crystalline preparations represent a heterogenous population of ribonucleoprotein complexes displaying a graduated activity and a variable RNA content. Free protein was not detected in the preparations.Fractionation of crystalline complexes in salt solutions of varying ionic strength and pH, followed by gel filtration on Sephadex G-200 led to the separation of two nucleoprotein fractions with very high specific activity. Fractions containing 30% RNA and 85% RNA respectively revealed a specific activity of 660–670 U/mg protein at 25°C.Pyruvate kinase form M_A was extracted from muscle homogenate with distilled water, purified to homogeneity and crystallized. It contained less than 0.2% RNA and had a specific activity of 270 U/mg. Active ribonucleoprotein complexes gave in double immunodiffusion test the precipitation bands with the anti-M_A sera at the same protein concentration of both antigens, M_B and M_A.Pyruvate kinase M_B with high activity is sensitive to treatment with RNase. Digestion with RNase for 10 min at 25°C diminished the initial specific activity to about one third. Similar residual activity was found in crystalline ribonucleo protein complexes with low RNA content (3.5–20% RNA) which are resistant to further inactivation by RNase.These results implicate the enhancement and control of pyruvate kinase activity by RNA bound to the enzyme.This work was supported by a grant from the Biochemical and Biophysical Committee of Polish Academy of Sciences.     

Postnatal changes in canine erythrocyte pyruvate kinase isozymes

The isozyme pattern of pyruvate kinase in canine erythrocytes changes following birth. These changes have been followed by electrophoretic, immunologic, and kinetic measurements of the isozymes. At birth, a mixture of isozymes is present consisting of the M₂ isozyme and hybrid molecules containing M₂ and R subunits. With increasing animal age, the content of M₂ subunits decreases and the content of R subunits increases. At 6 months of age, the isozyme pattern is indistinguishable from that of adult erythrocytes which contain only the R tetramer. We conclude that there is a switch in erythrocyte pyruvate kinase gene expression during the first 6 months of postnatal life. The existence of hybrid molecules during the switch indicates that both M₂ and R genes are expressed within each erythroid precursor cell. The developmental changes in erythrocyte pyruvate kinase are consistent with the role of this enzyme in the regulation of the oxygen-transport function of canine hemoglobin by 2,3-diphosphoglycerate in the postnatal period.This research was supported by Public Health Service Grant HD-10595.     

Tissue distribution of casein kinases

The activities of casein kinases 1 and 2 in cytosol fractions prepared from 12 different rat tissues were compared. Casein kinase activities were detected in all tissues examined. Total casein kinase activities of lung, spleen, testis, and thymus were much higher than those of skeletal muscle, cardiac muscle, and adrenal gland. When activities of casein kinases 1 and 2 partially purified from lung, spleen, testis, and thymus prepared from 5 rats were compared, both total and specific activities of these kinases in testis were higher than those in the other tissues. These results indicate that testis is the most suitable tissue in rats for large-scale purification of casein kinase 1 as well as casein kinase 2.     

Benzylidene ketal derivatives as M2 muscarinic receptor antagonists

Benzylidene ketal derivatives were investigated as selective M₂ receptor antagonists for the treatment of Alzheimer's disease. Compound 10 was discovered to have subnanomolar M₂ receptor affinity and 100-fold selectivity against other muscarinic receptors. Also, 10 demonstrated in vivo efficacy in rodent models of muscarinic activity and cognition.     

PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA

Abstract– The distribution of pyruvate kinase isozymes (EC 2.7.1.40) was examined in cells and tissues from the central and peripheral nervous system of the rat. Most tissues contain significant quantities of both the K₄ (fetal type) and M₄ (skeletal muscle type) isozymes plus tetrameric hybrids comprised of various combination of the type M and type K subunits. Retina, for example, contains a five-mem-bered hybrid set weighted toward K₄, while sciatic nerve and spinal cord have patterns very similar to that of adult brain, consisting predominantly of M₄ with small amounts of K₄ and K-M hybrids. This adult pattern is achieved by a gradual shift from a hybrid set dominated by K₄ in fetal life, to the pattern at birth at which time the two most prominent bands were M₄ and K₂M₂, and finally to the adult pattern by about 28 days after birth. Neurons and glial cells were isolated from rat and mouse brains at the various developmental levels. The pyruvate kinase isozyme patterns in the two cell types were similar to each other and to the patterns seen in whole brain homogenates at all ages, indicating similar rates of isozymic maturation in the two cell types. The correlation of maturation with pyruvate kinase isozyme patterns was further tested in cultures of malignant cell lines. A K-M hybrid set, weighted toward K₄, was seen in two clonal lines of mouse neuroblastoma under normal culture conditions. However, lowering the serum concentration in the culture medium or adding bromodeoxyuridine caused a shift in the patterns toward type M as the cells differentiated, mimicking in part the in vivo maturation of normal cells. On the other hand, a rapidly growing and poorly differentiated line of rat glioblastoma had only K₄ under all conditions examined.     

cAMP-dependent protein kinases, their subunits, and cAMP-binding protein from four sources: a comparison of physical characteristics determined by polyacrylamide gel electrophoresis

The physical characteristics of cAMP-dependent protein kinases and their, regulatory subunits from calf uterus, human uterus, human mammary tumor, and rat pituitary and of cAMP-binding protein from calf uterus were determined by quantitative polyacrylamide gel electrophoresis in buffers containing the detergent, Triton X-100. In the four tissues, protein kinases of either type A¹, with molecular weight (M_r) = 200,000, or type B, of M_r = 80,000, or both, previously described were found. Trivial charge isomerism, or size isomerism, exists within each of the two classes, Protein Kinase A and B. The protein kinase recombined from the regulatory and catalytic subunits is not significantly different from the crude or isolated protein kinase. Protein Kinases A and B exist each in either one of the isozyme forms I and II but these are not reflected in polyacrylamide gel electrophoresis at pH 10.2. Protein Kinase B appears to be a product of the partial proteolysis of Protein Kinase A. The regulatory subunits of Protein Kinases A from the four tissues are distinct from those of Protein Kinases B. No physical distinction exists between regulatory subunits derived from isozyme forms I and II. cAMP-Binding Proteins A and B are physically indistinguishable, by polyacrylamide gel electrophoresis at pH 10.2, from the regulatory subunits of Protein Kinases A and B, respectively.     

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

In the red cell incubated with ortho-[32P] phosphate, CaCl₂ and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl₂. In the presence of purified erythrocyte calmodulin, CaCl₂ and [γ-³²P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl₂. From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.     

Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf

1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.