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D L Blithe

Publications and source records attributed to D L Blithe.

At least 37 records · Page 2Linked to original sources
PubMed1989

Structure-kinetic relationships of choriogonadotropin and related molecules.

To assess how profound differences in carbohydrate and/or polypeptide structures affect parameters of plasma disappearance of glycoprotein hormones, we calculated and compared the initial volume of distribution, rate constants, and metabolic clearance rates of several highly purified human choriogonadotropin (hCG) analogues in monkeys. hCG, deglycosylated hCG, desialylated hCG, or core fragment of hCG-beta purified from pregnancy urine (beta-core) was administered as a rapid intravenous injection to adult male cynomolgus monkeys (n = 3/group). The metabolic clearance rates of deglycosylated hCG, beta-core fragment, and desialylated hCG were increased 15-, 47-, and 152-fold, respectively, over that of hCG. Their corresponding initial volumes of distribution, however, remained essentially unchanged compared with that of hCG and approximated the estimated plasma volume. In contrast, the fast and slow rate constants of plasma disappearance of the hCG analogues were increased as much as 18- and 23-fold, respectively, relative to those of hCG. These studies of structure-kinetic relationships in primates show that major carbohydrate and polypeptide modifications of a glycoprotein hormone cause profound changes in the rate constants of the disappearance curves without changes in the initial volume of distribution.

Animals↗
PubMed1989

Stimulation of testosterone production in the cynomolgus monkey in vivo by deglycosylated and desialylated human choriogonadotropin.

Modifications of carbohydrate structures of hCG, such as deglycosylation or desialylation, have been shown to reduce the biological activity of the hormone derivatives in vivo. We posed the question of whether deglycosylated hCG (dg-hCG) and desialylated hCG (ds-hCG) would behave as agonists at the LH/CG receptor in the primate in vivo, as this would bear on their potential clinical utility as LH/CG agonists or antagonists. Thus, we administered large doses (approximately 3 nmol) of highly purified dg-hCG, ds-hCG, hCG, or normal saline as a rapid iv injection to adult male cynomolgus monkeys (n = 3/group). Mean areas under the curves of plasma T over the first 6 h achieved with dg-hCG and ds-hCG were about 5-fold, significantly (P less than 0.05) greater than that in the saline controls and not significantly (P greater than 0.05) different from that in hCG-injected animals. Despite comparable plasma T responses in the first 6 h, mean plasma concentrations of ds-hCG, dg-hCG, and hCG differed dramatically among the groups. Plasma ds-hCG and dg-hCG levels were undetectable by 15 and 180 min, respectively, while the mean plasma hCG level was more than 2.10 nmol/L at 360 min. These data indicate that 1) dg-hCG is a full agonist at the LH/CG receptor in the primate in vivo, despite having minimal intrinsic activity in the rat Leydig cell adenyl cyclase assay and being able to near-completely antagonize hCG action therein; and 2) ds-hCG is a full agonist in the monkey in vivo, capable of stimulating a full testicular response over 6 h, despite being cleared from the circulation in 15 min. We conclude that the signal transduction system at the monkey LH/CG receptor is capable of achieving full steroidogenesis despite dramatically shortened exposure to stimulus or exposure to a stimulus with markedly reduced adenyl cyclase-stimulating activity in vitro.

Animals↗
PubMed1989

Carbohydrate composition of beta-core.

beta-Core is a major component of the hCG-related molecules found in pregnancy urine. We previously have purified the beta-core molecule and have deduced portions of its carbohydrate structure based on lectin binding data. In the present study we used recently developed technology to determine the carbohydrate composition of beta-core and hCG beta (CR119). For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both beta-core and hCG beta. hCG beta contained additional sugars consistent with the presence of four O-linked oligosaccharides. Compared to hCG beta, beta-core contained negligible sialic acid, galactose, or N-acetylgalactosamine. The compositional data suggest that beta-core does not contain N-acetylglucosamine at the nonreducing end of the molecule, whereas the trimannosyl-chitobiose core is apparently intact at both glycosylation sites, consistent with the ability of the molecule to bind to Concanavalin-A. Comparable fucose contents and abilities of beta-core and hCG beta to bind to Lens culinaris indicate a similar extent of fucosylation on the internal N-acetylglucosamine in both molecules. We propose that the N-linked oligosaccharides on beta-core closely resemble the underlying N-linked structures of hCG beta with the antennary sialic acid, galactose, and N-acetylglucosamine removed.

Carbohydrate Conformation↗
PubMed1989

Metabolic clearance rate and urinary clearance of purified beta-core.

We injected a highly purified preparation of the beta-core molecule, a fragment of hCG beta excreted in pregnancy urine, into five men and three women to determine its kinetic parameters, MCR, and urinary clearance. The beta-core molecule was distributed in an initial volume [1950 +/- 156 (mean +/- SEM) mL/m2 body surface area] approximately equal to the estimated plasma volume. Its disappearance was multiexponential on a semilogarithmic plot, with a rapid phase t1/2 of 3.5 +/- 0.7 min and a slow phase t1/2 of 22.4 +/- 4.2 min. The transit time (the mean time spent by a molecule of beta-core in transit) was 20.6 +/- 2.1 min. The MCR was 192.0 +/- 8.0 mL/min.m2 body surface area. About 5% of the injected dose of beta-core was excreted into the urine in the first 30 min after injection, and low levels of excretion persisted for up to 7 days. The urinary clearance rate of beta-core was 13.7 +/- 1.4 mL/min.m2, accounting for about 8% of the elimination of beta-core from the plasma. The beta-core immunoreactivity in serum and urine was characterized by gel filtration and three independent RIA systems to show that its properties were indistinguishable from those of the injected beta-core. Serum levels of beta-core in pregnant women were less than 0.2 ng/mL, while the amounts excreted in their urine were as much as 5 mg/day. Based on these clearance parameters of beta-core in normal subjects, less than 0.2% of the beta-core excreted in pregnancy urine is derived by urinary clearance of plasma beta-core. Therefore, more than 99% of the beta-core excreted in pregnancy urine is derived from beta-core in a compartment separate from plasma. In particular, these data indicate that there is relatively little placental secretion of beta-core into plasma and that placental secretion does not account for the vast majority of beta-core in pregnancy urine. These findings are consistent with previous data that point to renal parenchymal degradation of hCG and hCG beta as the major source of urinary beta-core in pregnancy.

Adult↗
PubMed1988

Purification of beta-core fragment from pregnancy urine and demonstration that its carbohydrate moieties differ from those of native human chorionic gonadotropin-beta.

Pregnancy urine contains large quantities of hCG, free beta-subunit, free alpha-subunit, and a population of fragments of beta-subunit known as beta-core. This beta-core population, which can account for as much as 70% of the total beta-immunoreactivity in pregnancy urine, is of interest as both a normal metabolite of pregnancy and a potential marker for malignancy. We have purified the beta-core fragment from pregnancy urine (P-core) and have characterized it with respect to size and carbohydrate composition. P-Core was purified by chromatography on Sephadex G-100, Concanavalin A (Con A)-Sepharose, DEAE-Sephacel, and Sephadex G-75 (superfine). The purified P-core has an apparent mol wt of 17,500 and 17,000, as determined by gel filtration on Sephadex G-75 (superfine) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, respectively. The sialic acid content of P-core was assayed chemically and was less than 0.07 mumol sialic acid/mumole P-core. For comparison to P-core, we have prepared a trypsin fragment of beta-subunit that retains the beta-core immunological determinant recognized by SB6 antiserum and lacks the carboxy-terminal immunological determinant. We have designated this beta-core molecule as T-core (tryptic fragment of beta-subunit) to distinguish it from the beta-core molecule that we have purified from pregnancy urine (i.e. P-core). Most of the P-core and T-core molecules bind to Con A (84% and 86%, respectively). The Con A-bound material was used for subsequent characterizations. Neither P-core nor T-core binds to DEAE using conditions under which intact hCG beta binds to DEAE. A variety of agarose-bound lectins were used to further investigate the carbohydrate nature of the Con A-bound P-core and T-core molecules. The lectin binding data indicate that the antennae on P-core do not contain appreciable sialic acid or galactose, in contrast to the antennae on T-core, which contain both. We conclude that P-core, the naturally occurring beta-core fragment in pregnancy, has been processed to a form in which nearly all of the sialic acid and galactose residues are removed, but the Con A-binding site (consisting of the core sugars) and most of the core fucose are retained.

Carbohydrate Conformation↗
PubMed1988

A radioimmunoassay for the core fragment of the human chorionic gonadotropin beta-subunit.

We developed a RIA for the beta-core fragment of hCG that is excreted in the urine of pregnant women and some patients with cancer. We purified beta-core from crude commercial preparations of hCG (of which beta-core is a major constituent) derived from pregnancy urine and used this purified beta-core material to immunize rabbits. One antiserum (RW25) was particularly useful in that a RIA with purified beta-core as both radioligand and reference preparation had high sensitivity for beta-core detection and low cross-reactivity with other hCG-related molecules and glycoprotein hormones. The cross-reactivities of purified hCG (CR125), hCG beta (CR125), and hCG alpha (CR125) preparations were in each instance less than 3 x 10(-3) (wt/wt). The cross-reactivities of purified pituitary glycoprotein hormones were in each instance less than 2 x 10(-4) (wt/wt). Using the RW25 RIA, virtually all beta-core immunoreactivity in pregnancy urine eluted from Sephadex G-100 in a position coincident with that of purified beta-core. Urine from men and nonpregnant women contained very low levels of beta-core immunoreactivity (less than 6.5 micrograms/L), while urine from pregnant women and patients with testicular cancer or other neoplasms had levels of beta-core immunoreactivity ranging as high as 26,000 micrograms/L. We conclude that the improved specificity of our beta-core RIA will facilitate studies of the physiology and cancer biology of beta-core molecules.

Adolescent↗
PubMed1988

Beta-core fragments are contaminants of the World Health Organization Reference Preparations of human choriogonadotrophin and its alpha-subunit.

Highly purified preparations of human choriogonadotrophin (hCG), hCG alpha and hCG beta, including those preparations which are being distributed by the World Health Organization as International Standards, cross-reacted in a new radioimmunoassay with increased relative specificity for the beta-core fragment of hCG. A major portion of the beta-core immunoreactivity in the hCG and hCG alpha preparations eluted from Sephadex G-100 in a position (approximate apparent molecular size 15,000-18,000) corresponding to that of purified beta-core fragment prepared from pregnancy urine. However, in the case of hCG beta-subunit preparations, virtually all of the beta-core cross-reacting material eluted from Sephadex G-100 in the same fractions as the native hCG beta-subunit. Quantitatively, the cross-reacting beta-core material accounts for less than 1% (w/w) of the total hCG or subunit immunoreactivity, as measured by conventional radioimmunoassays. The presence of the beta-core fragments as discrete molecular components of the hCG and hCG alpha preparations should be borne in mind when these preparations are used to calibrate new radioimmunoassays for hCG-related molecules.

Chorionic Gonadotropin↗
PubMed1987

Similarity of the clearance rates of free alpha-subunit and alpha-subunit dissociated from intact human chorionic gonadotropin, despite differences in sialic acid contents.

It is well known that the MCR of proteins can be influenced by their carbohydrate structure; i.e. the presence of terminal galactose on proteins results in uptake by hepatic receptors for galactose-terminated glycoproteins. Thus, a protein with its galactose residues covered or removed exhibits a far longer half life in plasma than one with its galactose residues exposed. The free alpha-subunit of human CG (hCG) has been shown to have a different carbohydrate composition than does the alpha-subunit dissociated from the intact hormone. In our laboratory, analysis of alpha-subunits isolated from pregnancy urine indicated that the alpha-subunit dissociated from hCG (hCG alpha) contains primarily monosialylated oligosaccharides, whereas the free alpha-subunit contains more than one sialic acid per oligosaccharide. This difference in the degree of sialylation prompted us to examine the clearance rates of these two subunits. Accordingly, free alpha and hCG alpha were purified by affinity chromatography and labeled with 125I. The labeled subunits were injected iv into rats and serum samples were removed at various time intervals over a 2-h period. The amount of [125I]alpha-subunit remaining in the serum was determined by immunoprecipitation using an antiserum to hCG alpha. The disappearance curves for the two subunits were indistinguishable and could be analyzed by a biexponential model. The t 1/2 of the faster component was 5 min, while the t 1/2 of the slower component was 74 min. In order to determine whether or not terminal galactose was present on either the hCG alpha or the free alpha-subunit, the labeled molecules were subjected to lectin column chromatography using Ricin or peanut lectin linked to agarose. Both of these lectins bind galactose but have different specificities with respect to the penultimate sugar. Both subunit preparations contained only minor amounts of material which could bind or either lectin. However, after desialylation, both hCG alpha and free alpha bound extensively to Ricin, indicating the presence of penultimate galactose residues in both. We conclude that terminal galactose residues are not present on the oligosaccharides of either hCG alpha or free alpha-subunits, and that the difference observed in the sialic acid contents of the two subunits does not affect their rates of clearance.

Animals↗
PubMed1986

Inactivation of follicle-stimulating hormone by a factor in a bovine serum albumin preparation.

Bovine serum albumin (BSA) preparations are commonly employed as "carrier" or "protective" proteins in the solutions used to dissolve gonadotropin preparations. The present report describes a BSA preparation that was found to contain a factor that inactivated follicle-stimulating hormone (FSH). Four different BSA preparations (designated BSA1, BSA2a, BSA2b, BSA3) were studied. The FSH preparation (NIH-FSH-S16) was dissolved in 0.15 M NaCl, containing the various BSA preparations. The FSH solutions were injected subcutaneously, twice daily, for 5 days into hypophysectomized immature female rats bearing estrogen capsules. Twenty-four hours after the last injection, the rats were decapitated, and the ovaries were removed, trimmed and weighed. The FSH preparation produced ovarian weight gain when BSA1, BSA2b, or BSA3 was used, but not when BSA2a was used in the vehicle. In animals injected with the FSH dissolved in BSA1 vehicle and injected at a separate site with BSA2a solution, the FSH preparation was fully active, which indicates that contact of the BSA2a preparation with FSH was required for the inactivating factor to be operant. Indeed, after incubation in BSA2a solution, the radiolabeled FSH preparation exhibited a slight decrease in apparent molecular size when chromatographed on a Sephadex G-100 column. This result suggests that the BSA2a preparation contained a factor that may have inhibited FSH by degrading it.

Animals↗
PubMed1986

Inhibition of follicle-stimulating hormone/diethylstilbestrol-stimulated ovarian growth by extracts of pregnancy urine.

We devised an in vivo biological assay for ovarian growth inhibiting activity to examine extracts of human pregnancy urine for the presence of ovarian growth inhibiting factor. Diethylstilbestrol (DES) capsules were implanted sc in immature hypophysectomized female rats; FSH was injected sc with or without test substance for 5 days. Rats with unstimulated ovaries were implanted with blank capsules and given the vehicle without FSH. Twenty four hours after the last injection, the ovaries were removed and weighed. The ovarian growth inhibition of the ovarian weight gain achieved in rats treated with DES and FSH. Crude commercial human CG (hCG) preparations, extracted from pregnancy urine, were chromatographed on Sephadex G-100, and the fractions were tested for ovarian growth inhibiting activity. The peak of ovarian growth inhibiting activity was found in fractions eluting from the column in the mol wt range of 12,000-20,000. Ovarian growth inhibiting activity was heat sensitive, not extracted by ether, and precipitated by acetone. Ovarian growth inhibiting activity was stable in acid at pH 2, but was inactivated by digestion with trypsin. The ovarian growth inhibiting activity was purified by chromatography on Concanavalin A-Sepharose and diethylaminoethyl-Sephacel. The active material contained hCG alpha, hCG beta, and beta-carboxyterminal peptide-immunoreactivity and its inhibiting activity could be removed from solution by immunoadsorption with antisera specific for hCG beta. The ovarian growth inhibiting activity was further purified on an anti-hCG alpha-immunoglobulin G affinity column. The activity was eluted from the affinity column at low pH, and eluted material contained all of the immunodeterminants of hCG. Virtually identical dose-response curves of ovarian inhibition were obtained using equivalent doses of beta-carboxyterminal peptide immunoreactivity of purified inhibitor and purified hCG (CR123). The inhibiting activity reached plateau of 80-90% at doses of 50-100 ng hCG/rat. Upon rechromatography on Sephadex G-100, the ovarian growth activity that was pooled from fractions corresponding to the 12,000-20,000 mol wt range was recovered in fractions corresponding to the elution position of hCG. We conclude that the low mol wt inhibiting activity observed in the crude pregnancy extracts is due to hCG and that hCG is a very potent inhibitor of FSH/DES stimulation of ovarian growth.

Acetone↗
PubMed1985

Variations in the oligosaccharides on free and combined alpha-subunits of human choriogonadotropin in pregnancy.

The glycoprotein hormone hCG and a free alpha-subunit are secreted by trophoblastic cells during pregnancy. We have purified the alpha-subunit of hCG and the free alpha-subunit population from pregnancy urine. Dissociation of hCG with 10 M urea at 37 C, followed by chromatography on DEAE-Sephacel, resulted in separation of alpha- from beta-subunit, as hCG alpha does not bind to DEAE in the presence of urea, while beta-subunit does bind. Similar treatment of the free alpha population resulted in fractionation into two populations, a nonbinding fraction of free alpha and a population which was retained by DEAE in the presence of urea (free alpha 2). The three populations, hCG alpha, free alpha, and free alpha 2, were further purified by affinity chromatography using anti-alpha antisera linked to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed that hCG alpha consisted of a single component with an apparent mol wt of 22,000, while free alpha and free alpha 2 consisted of multiple components. Radioactive labeling of sialic acid by limited periodate oxidation and NaB[3H]4 reduction resulted in a higher specific activity for free alpha than for hCG alpha, suggesting that free alpha contains more sialic acid per immunoreactive molecule than does alpha dissociated from hCG. [3H]hCG alpha, but not 3H-labeled free alpha, was able to combine with native hCG beta-subunit. The oligosaccharide moieties were released from the different labeled subunits by alkaline hydrolysis and then compared with respect to Concanavalin A (ConA)-binding and DEAE-binding properties. Most of the oligosaccharides from dissociated hCG alpha bound to ConA-Sepharose (72%), while less material from free alpha (40%) and even less from free alpha 2 (25%) were capable of binding to ConA. DEAE chromatography of the oligosaccharides suggested that hCG alpha contained primarily monosialylated forms (greater than 60%), while free alpha and alpha 2 contained primarily (greater than 70%) di- and trisialylated forms. Thus, the ConA and DEAE binding data indicated that the oligosaccharide contents of free alpha and free alpha 2 were quite different from that of hCG alpha. These results also suggest that some of the oligosaccharide structures contained on hCG alpha and most of those on free alpha and free alpha 2 differ substantially from the structures that have been previously described.

Carbohydrate Sequence↗
PubMed1983

Characterization of the insulin receptor kinase purified from human placental membranes.

The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.

Animals↗
PubMed1983

Tyrosine-specific protein kinase activity is associated with the purified insulin receptor.

Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [gamma-32P]ATP in the presence of MnCl2 (2 mM) and analyzed in NaDodSO4/acrylamide gel electrophoresis, only the Mr 95,000 band was labeled. Preincubation with several concentrations of insulin increased the 32P incorporation into this peptide in dose-dependent fashion, whereas insulin-like growth factors were approximately equal to 2% as potent and epidermal growth factor had little or no effect, consistent with their known affinities for the insulin receptor. Insulin stimulation of phosphorylation of the Mr 95,000 subunit of the receptor was observed also in immunoprecipitates of this highly purified insulin receptor by anti-insulin-receptor antibodies. Phosphoamino acid determination revealed only phosphotyrosine in both the basal and insulin-stimulated states. These data suggest that a tyrosine-specific protein kinase activity is closely associated with insulin receptor, and this may be important in the signal transmission required for insulin action.

Female↗
PubMed1982

Similarity in the bound carbohydrate groups of glycoproteins from cells of several vertebrate classes.

The carbohydrate groups of the glycoproteins of human, hamster, chick, reptile and fish cells growing in culture have been fractionated in succession according to size (Sephadex G-50), affinity for concanavalin A, charge (DEAE-Sephadex) and by thin-layer chromatography. It was found that despite the complexity of the array of separable glycopeptides in each type of cell, most of these structures seemed to be common to all of the cells. This suggests that they have existed in a relatively stable state for several hundreds of millions of years throughout the evolution of the vertebrates.

Animals↗
PubMed1982

Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor.

We have identified a tyrosine kinase activity present in tumors which were raised in rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This kinase phosphorylates tyrosine on the heavy chain of IgG from tumor-bearing rabbit (TBR) sera specific for the src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we have purified this kinase over 7200-fold. The purification procedure involves detergent extraction of tumors followed by sequential column chromatography on hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, an affinity column prepared from TBR-sera, and Sephacryl S-200. The IgG kinase activity behaves as a molecule of apparent Mr = 54,000 on Sephacryl S-200 molecular sieve chromatography. Analysis of the Sephacryl fractions by SDS-PAGE indicates that a major Coomassie blue-stained band with an apparent Mr = 54,000 (p54), co-elutes with the peak of kinase activity. From 600 g of tumors, approximately 200 micrograms of p54 are obtained. We have four types of evidence which show that p54 is related to pp60src. 1) Purified p54 is capable of undergoing endogenous phosphorylation in the presence of [gamma-32P]ATP producing a 32P-labeled pp54 polypeptide which is specifically immunoprecipitated by TBR-sera and contains only phosphotyrosine. 2) Purified p54 competes with 32P-labeled pp60src for binding to TBR-IgG, indicating a degree of purification over starting material which agrees very well with the results obtained by the IgG kinase assay. 3) V8 protease digestion of pp60src and p54 suggests that they share a common 26,000 fragment. 4) Antibodies to partially purified p54 specifically precipitate pp60src from Rous sarcoma virus-transformed chicken cells.

Animals↗
PubMed1982

Properties of the src kinase purified from Rous sarcoma virus-induced rat tumors.

We recently described a method for the purification of a protein kinase related to pp60src from Rous sarcoma virus-induced rat tumors (Blithe, D. L., Richert, N. D., and Pastan, I. H. (1982) J. Biol. Chem. 257, 7135-7142). In this report, we describe some of the properties of the 7200-fold purified enzyme. The purified kinase phosphorylates casein, vinculin, actin, and histone H2B, but not bovine serum albumin or ovalbumin. Protein substrates are phosphorylated exclusively on tyrosine residues. Casein was used as a substrate for more detailed analysis. The phosphorylation reaction proceeds at a linear rate for at least 40 min at 22 degrees C. Maximum enzyme activity occurs at pH 6.5 to pH 6.8 and requires the presence of either Mg2+ (5 to 10 mM) or Mn2+ (1 to 5 mM). The Km for ATP is 30 microM and the Vmax 0.03 mumol/min/mg using 0.4 mg/ml of casein as a substrate. The enzyme utilizes ATP or dATP, but not GTP as a phosphate donor in the reaction. The enzyme is inhibited by adenosine and deoxyadenosine and by their corresponding mono- and diphosphates. No inhibition of enzyme activity is observed with adenine, GTP, UTP, TTP, or CTP. The enzyme is very sensitive to increased ionic strength. Addition of 0.1 M KCl, 0.1 M NaCl, 50 mM KPO4, or 50 mM NaPO4 inhibited casein phosphorylation by 90 to 95%. Analysis of the products of the phosphorylation reaction by thin layer chromatography revealed that the src kinase phosphorylates glycerol in addition to casein or the enzyme itself.

Animals↗