Characterization of heparin-binding endothelial mitogen(s) produced by the ovine endometrium during early pregnancy.

To characterize mitogenic factors produced by ovine endometrium during early pregnancy, endometrial explant-conditioned media (ECM) were obtained from ewes on day 12, 18, 24, or 30 after mating. These ECM contained mitogenic activity for both endothelial and 3T3 cells across all days. The endothelial mitogenic activity was greatest on day 24, whereas mitogenic activity for 3T3 cells did not differ across days. By ultrafiltration, ion exchange, and heparin-affinity chromatography, the endothelial mitogenic activity was found to have a molecular mass greater than 100 kDa, to be anionic, and to be heparin binding, respectively. Three peaks of endothelial mitogenic activity were recovered from heparin-affinity chromatography. The major peak, H3, was mitogenic for endothelial but not for 3T3 cells. H3 was further purified, and the single peak of heparin-binding activity, designated H3b, represented a 681-fold purification of endothelial mitogenic activity from endometrial ECM. H3 and H3b were heat labile and trypsin sensitive, and their biological activity was heparin enhanced. The majority of the endothelial mitogenic activity was immunoneutralized by antibodies against acidic and basic FGF. Nevertheless, we were unable to detect bFGF in H3 or H3b by Western immunoblot analysis. Thus, in this study we have extended our previous observations and demonstrated that (i) during early pregnancy the ovine endometrium produces mitogenic activity for both endothelial and 3T3 cells, (ii) the endothelial mitogenic activity is greatest on day 24 after mating. which corresponds with the onset of endometrial vascular growth, and (iii) the major endothelial mitogen has a high affinity for heparin, and although it is immunologically related to FGF, it differs from known FGF in its apparent molecular size and biological activities.

Stimulation of phosphorylase phosphatase activity of protein phosphatase 2A1 by protamine is ionic strength dependent and involves interaction of protamine with both substrate and enzyme.

The effects of protamine on the phosphorylase phosphatase activity of porcine cardiac protein phosphatase 2A1 (PP2A1) were complex and ionic strength dependent. Under ionic strength conditions that protamine activation was optimal, activation of PP2A1 by either dilution or heparin was prevented. A time-dependent deactivation of the protamine-stimulated phosphatase activity was observed when PP2A1 was preincubated with protamine. Protamine forms a very tight association with phosphorylase a, which is optimal at a 1:1 protamine:phosphorylase a monomer molar ratio. Protamine activation of PP2A1 activity, however, is not substrate-directed since the basic polypeptide did not stimulate either the activity of the catalytic subunit or trypsinolysis of [32P]phosphorylase a. The interaction of protamine with phosphorylase a does not apparently involve the phosphorylation site in the protein substrate (ser 14). The activation of PP2A1 by protamine is proposed to involve part of the basic polypeptide, not associated with phosphorylase a monomer, interacting with the regulatory and/or the catalytic subunit(s) of the phosphatase. A minimal model for the activation of PP2A1 by protamine was tested kinetically. In this model, free PP2A1 binds with decreasing affinities to the protamine:phosphorylase a complex, free phosphorylase a, and free protamine. Protamine decreases the K(m) of PP2A1 for the phosphorylase a monomer 5-fold and increases the Vmax 17-fold. Interaction of free protamine with PP2A1 inhibits the phosphatase activity.

A continuous spectrophotometric assay for phosphorylase kinase.

A continuous spectrophotometric assay for the determination of the initial rate of the phosphorylase kinase catalyzed reaction at pH 7.0 is presented. The assay incorporates two coupling enzyme systems: (a) recombinant rabbit skeletal muscle type 1 protein phosphatase catalytic subunit which dephosphorylates the phosphorylase a product of the phosphorylase kinase reaction, and (b) the system of Webb (Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992), which uses purine nucleoside phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for the quantitation of the resultant inorganic phosphate. The effects of reaction components on the enzyme activities were studied. The system was standardized and validated. The continuous coupled enzyme system was used for the kinetic analysis of nonactivated phosphorylase kinase at pH 7.0. Km and kcat values of 15.36 +/- 0.2 microM (phosphorylase b monomer) and 21 +/- 1.12 s-1, respectively, were determined.

A continuous spectrophotometric assay for protein phosphatases.

A continuous spectrophotometric assay for the determination of protein phosphatase activity is presented. The assay incorporates the coupled enzyme system of Webb (M. R. Webb, 1992, Proc. Natl. Acad. Sci. USA 89, 4884-4887), which used purine nucleoside phosphorylase and the chromophoric substrate 7-methyl-6-thioguanosine for the quantitation of inorganic phosphate. The assay is exemplified and validated here for the phosphorylase phosphatase activity of protamine-stimulated protein phosphatase 2A1 (PP-2A1). The effects of reaction components on the activities of both PP-2A1 and purine nucleoside phosphorylase were studied. The application of the coupled assay system to kinetic analysis of the phosphorylase phosphatase activity of PP-2A1 and to the assay of the catalytic subunits of type 1 and 2A protein phosphatases and a recombinant type 1 catalytic subunit is demonstrated. The applicability of this coupled enzyme system to the assay of other protein phosphatases is discussed.

Initial characterization of mitogenic activity of ovine corpora lutea from early pregnancy.

To characterize angiogenic factors produced by ovine corpora lutea (CL) during early pregnancy, two experiments were performed. In Experiment 1, luteal explants from days 12, 18, 24, and 30 (n=4 ewes/day) after mating were incubated in serum-free medium for 6 h. Luteal-conditioned media (LCM) were evaluated for their ability to stimulate proliferation of endothelial and 3T3 cells, as well as migration of endothelial cells. Pools of the LCM (one pool/day) then were characterized biochemically. In Experiment 2, two pools of LCM from days 24 of pregnancy were evaluated for their effects on endothelial cell, 3T3 cell, and ovine luteal cell proliferation. These pools of LCM then were concentrated by ultrafiltration and subjected to heparin-agarose affinity chromatography with salt gradient (0-4 M NaCl in buffer) elution, and fractions were evaluated for mitogenic activity for endothelial and 3T3 cells. The resulting five peaks of mitogenic activity from heparin-agarose chromatography were characterized biochemically. The five peaks of mitogenic activity were further purified by using chromatography, then were concentrated and subjected to SDS-PAGE and Western analysis for FGF-2. Ovine CL from each day of early pregnancy secreted mitogens (P<0.05) for endothelial (285 +/- 8% of unconditioned media controls) and 3T3 (142 +/- 7%) cells as well as factors which stimulated migration of endothelial cell (153 +/- 8% of controls). LCM pool from day 24 of pregnancy also stimulated (P<0.05) proliferation of ovine luteal cells in a dose-dependent manner. In Experiment 1, mitogenic activity for endothelial cells was greater than 100 kDa, heat-labile, trypsin-sensitive and bound to DEAE-Sephacel and heparin-agarose columns, but not to a CM-Sepharose column. Antibody against FGF-1 did not affect mitogenic activity of LCM for endothelial and 3T3 cells, whereas treatment with FGF-2 antibody decreased (P<0.05) mitogenic activity of LCM for both endothelial and 3T3 cells. In Experiment 2, heparin-agarose affinity chromatography resolved five peaks of mitogenic activity: a non-heparin-binding peak that was specific for 3T3 cells, three heparin-binding peaks that were specific for endothelial cells, and one heparin-binding peak that was specific for 3T3 cells. In Experiment 2, heparin-, heat-, or trypsin-treatment and immunoneutralization with FGF-1 or FGF-2 antibodies influenced mitogenic activity of all of the peaks. Whereas SDS-PAGE demonstrated several bands of protein within each peak, Western analysis was unable to detect the presence of FGF-2 in any of the heparin-binding peaks. These data demonstrate that ovine CL from early pregnancy produce mitogenic factors that can be resolved into 5 separate peaks of activity with differing affinities for heparin. These data also indicate that the endothelial mitogens produced by CL of early pregnancy are immunologically related to, but biochemically distinct from FGF-2. Mitogens for endothelial and other cells likely play a role in regulation of luteal function during early pregnancy in sheep.

Purification and characterization of bovine heart glycogen synthase kinase-3.

Glycogen Synthase Kinase-3 (GSK-3) was isolated from bovine heart tissue extracts by a procedure involving ammonium sulfate fractionation, followed by chromatography on phosphocellulose, Cibacron blue 3GA-agarose, DEAE-Sephacel, CM-Sepharose, heparin-agarose, myelin basic protein-Sepharose, and LiChrospher 1000 C00-. GSK-3 was identified by its activation of protein phosphatase-1i (PP-1i). The purified enzyme had a specific activity of 25,500 units of protein phosphatase-1i activated/mg protein. The enzyme is an asymmetric monomeric protein of 53 kDa. The molecular size and retention of activity after autophosphorylation indicated that the isolated enzyme was the GSK-3 alpha-isoform.

Mitogenic factors of corpora lutea.

The mammalian corpus luteum (CL), which plays a central role in the reproductive process because of its production of hormones such as progesterone, appears to be an exceptionally dynamic organ. Its rate of growth and development are extremely rapid and, even when the CL is functionally mature, its rate of cell turnover remains relatively high. Associated with this high rate of cell turnover, the mature CL receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Although numerous growth factors have been identified in luteal tissue, their role in growth and differentiation of this dynamic organ remains unclear. Recently, while attempting to identify mitogenic factors of ovine and bovine CL, we have found that they produce several mitogens during the estrous cycle as well as pregnancy. The majority of these luteal-derived mitogenic factors are heparin-binding, and although some may represent previously identified factors, several appear to be novel heparin-binding growth factors. Isolation and purification of mitogenic factors produced by the CL will enable us to determine their roles in luteal growth, development and differentiated function, which will contribute to our understanding not only of the regulation of fertility but also of tissue growth and development in general.

Initial characterization of endothelial mitogens produced by bovine corpora lutea from the estrous cycle.

To further characterize mitogenic factor(s) present in luteal extracts or luteal explant conditioned media (LCM), bovine corpora lutea (CL) were homogenized or incubated in explant culture, respectively. After evaluation of luteal extracts and LCM by using an endothelial cell proliferation bioassay, mitogenic activity was characterized by immunoneutralization with antibodies against heparin-binding (fibroblast) growth factor (HBGF) 1 or 2. LCM also were subjected to ultrafiltration, as well as anion-exchange, cation-exchange, and heparin-affinity chromatography. The presence of HBGF-2 in LCM also was evaluated by using a dot immunoblot assay. Extracts of luteal tissues and LCM stimulated (P < 0.05) proliferation of endothelial cells in a dose-dependent manner. Mitogenic activity of luteal extracts and LCM was decreased (P < 0.05) by treatment with specific antibodies against HBGF-2 or HBGF-1. LCM also contained immunoreactive HBGF-2. The mitogenic activity bound to anion exchangers, phenyl-Sepharose, and heparin-agarose, but not to cation exchangers, indicating that endothelial mitogenic activity is anionic at neutral pH, has some hydrophobic characteristics, and belongs to the HBGF family of proteins. Following ultrafiltration, endothelial mitogenic activity was retained by membranes having a 30,000 or 100,000 molecular weight cutoff. In addition, LCM was resolved into four peaks of heparin-binding endothelial mitogenic activity, each with a different affinity for heparin. These data demonstrate that bovine CL contain and produce endothelial mitogens of large molecular size, which may be important regulators of luteal function. These endothelial mitogens are heparin-binding and anionic at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Hierarchical regulation by casein kinases I and II of the activation of protein phosphatase-1i by glycogen synthase kinase-3 is ionic strength dependent.

The roles of casein kinases I and II in the activation of protein phosphatase-1i (PP-1i) by glycogen synthase kinase-3 (GSK-3) were studied using enzyme preparations from porcine heart. PP-1i was activated by GSK-3 and the levels of activation achieved decreased by increasing the ionic strength (0-0.2 M KCl) in the incubation mixtures. At low ionic strength (no KCl added) casein kinase II increased the rate of activation of PP-1i by GSK-3 and the activation proceeded to a slightly greater extent (110-120%) than that obtained by GSK-3 alone. In the presence of 0.14 M KCl only a partial activation of PP-1i by GSK-3 was observed, but when casein kinase II was also added activation was restored to levels observed when PP-1i was activated by GSK-3 in the absence of salt. This effect was shown to be dependent on the concentration of casein kinase II. These results would imply that at low ionic strength casein kinase II and GSK-3 synergistically activate PP-1i as has been previously reported for the rabbit skeletal muscle enzyme (DePaoli-Roach, A. A., J. Biol. Chem. 259, 12144-12152, 1984), whereas, at physiological ionic strength, casein kinase II action may be obligatory for GSK-3 activity. Similar results were obtained when casein kinase I replaced casein kinase II.

Kinetic evaluation of substrate-dependent origin of the lag phase in soybean lipoxygenase-1 catalyzed reactions.

We have measured, under identical conditions, the time courses for the native lipoxygenase (Fe2+ form)-catalyzed conversion of linoleic acid into 13-hydroperoxy-9,11-octadecadienoic acid (HPOD) and the oxidation of the Fe2+ form of enzyme to the Fe3+ form (in 0.1 M borate buffer, pH 10.0, at 25 degrees C) using a stopped-flow spectrophoto/fluorometer. The experimental results clearly demonstrate that the time course for the appearance of the reaction product is much shorter than that for the conversion of E-Fe2+ to E-Fe3+; the latter process involves a pronounced lag phase whereas the former does not. This suggests that the Fe2+ form of the enzyme is also catalytically active and that the origin of the lag phase is not intrinsic to the oxidation of the enzyme bound iron cofactor. When the Fe3+ form of the enzyme is utilized to investigate the time course of product formation, the lag phase was observed at substrate concentrations higher than 20 microM. The magnitude of this lag phase increases with the increases with the increase in the initial concentration of the substrate (at least up to the range where substrate is not dimerized) and decreases in the presence of increasing concentrations of HPOD (exogenously added to the reaction mixture). No lag phase is evident at substrate concentrations in the range of 10 microM or less. We have examined the effects of varied concentrations of substrate and product on the initial rates of the lipoxygenase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of porcine heart type 2A protein phosphatases.

Protein phosphatases 2A1 and 2A2 were isolated from porcine heart tissue extracts by precipitation at pH 5.0 and separated by chromatography on DEAE-Sephacel. Phosphatase 2A1 was then purified to apparent homogeneity by chromatography on phenyl-Sepharose, aminohexyl-Sepharose, Sephacryl S-300, and L-tyrosine-agarose. Phosphatase 2A2 was purified to apparent homogeneity by chromatography on phenyl-Sepharose, DEAE-Sephacel, aminohexyl-Sepharose and L-tyrosine-agarose. Purified phosphatases 2A1 and 2A2 had specific activities of 2200 and 2710 nanomoles of phosphate released from phosphorylase a/mg protein, respectively. The apparent molecular weights of phosphatases 2A1 and 2A2 on gel filtration were 155 and 105 kDa, respectively. Both enzymes contain 70 and 37 kDa subunits and 2A1 also contains a 57 kDa subunit. The 37 kDa catalytic subunit (2Ac) was obtained from the purified phosphatases by treatment with room temperature ethanol followed by sucrose density gradient centrifugation or gel filtration chromatography.

Purification of porcine heart latent protein phosphatase Fc.M.

Latent protein phosphatase, Fc.M, was purified from porcine heart extracts by a procedure involving precipitation at pH 5.0, DEAE-Sephacel chromatography, ammonium sulfate fractionation, chromatography on phenyl-Sepharose, Biogel-A 0.5m and poly-L-lysine-agarose. The purified enzyme had a specific activity of 12,200 nanomoles of phosphate released from phosphorylase a/mg protein when assayed following activation by pretreatment with Mn++ and trypsin in the presence of 0.2 M NaCl. The enzyme is a heterodimer of 66 kDa composed of a catalytic (37 kDa) and a modulator (31 kDa) subunit.

Effects of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on porcine heart protein phosphatase 2A.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate are apparent noncompetitive inhibitors of porcine protein phosphatase 2A2 having Ki values of 0.38 and 0.56 mM, respectively. The inhibitory effects were on the catalytic subunit and were not substrate directed. In addition, fructose 2,6-bisphosphate caused a time-dependent inactivation of phosphatase activity toward phosphorylase a. This inactivation was antagonized by MnCl2. The fructose 2,6-bisphosphate-inactivated enzyme had increased p-nitrophenyl phosphate phosphatase activity. These effects are similar to the known effects of ATP on type 2A phosphatases.

Production of mitogenic factor(s) by ovine corpora lutea throughout the estrous cycle.

To evaluate secretion of mitogenic factors by ovine corpora lutea (CL) at several stages of development, luteal explants from days 5 (n = 12 ewes), 10 (n = 6 ewes), and 15 (n = 6 ewes) of the estrous cycle were incubated in serum-free medium for 24 h. Luteal-conditioned media (LCM) were evaluated for their ability to stimulate proliferation of endothelial, BALB/3T3, and ovarian granulosa cells. After mitogenic activity of LCM from individual animals was evaluated, pools of LCM from each day of the estrous cycle were subjected to anion-exchange, cation-exchange, and heparin-affinity chromatography to characterize mitogenic activity. Pools of LCM also were utilized for ultrafiltration, heat-treatment, trypsin-treatment, and immunoneutralization studies. Results demonstrated that ovine CL secrete mitogenic activity that stimulates (P less than 0.01) proliferation of endothelial (135.7 +/- 5.3% of control) and granulosa cells (188.9 +/- 2.9%) but not 3T3 (103.2 +/- 2.5%) cells. Differences between stages of luteal development were not observed. The mitogenic activity bound to diethylaminoethyl-Sephacel and heparin-agarose, but not to carboxymethyl-Sepharose, indicating that ovine luteal mitogenic factor(s) is anionic and may belong to the heparin-binding growth factor (HBGF) family. In addition, the mitogenic activity was heat-labile, trypsin-sensitive, and appeared to have a M(r) greater than 100,000. Mitogenic activity for endothelial cells was partially neutralized with a specific antibody against HBGF-1 and was completely abolished with a specific antibody against HBGF-2. Moreover, HBGF-1 and HBGF-2 were immunolocalized in histological sections of CL from days 5 (n = 5 ewes), 10 (n = 5 ewes), and 15 (n = 5 ewes) after estrus. These findings are the first report of a major mitogenic factor(s) produced by cyclic ovine CL and indicate this factor is an HBGF-2-like molecule.

Angiogenesis in the female reproductive system.

In adult tissues, capillary growth (angiogenesis) occurs normally during tissue repair, such as in healing of wounds and fractures. Rampant capillary growth is associated with various pathological conditions, including tumor growth, retinopathies, hemangiomas, fibroses and rheumatoid arthritis. The female reproductive organs (i.e., ovary, uterus, and placenta) exhibit dynamic, periodic growth and regression accompanied by equally dramatic changes in rates of blood flow. It is not surprising, therefore, that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Thus, the female reproductive system provides a unique model for studying regulation of angiogenesis during growth and differentiation of normal adult tissues. Ovarian, uterine, and placental tissues recently have been shown to contain and produce angiogenic and anti-angiogenic factors. This review discusses the current state of knowledge regarding angiogenic processes and their regulation in female reproductive tissues. In addition, implications of this research for regulation of fertility as well as for control of angiogenesis in other normal and pathological processes are discussed.

Evidence for an inactive plasma membrane-associated precursor of active cytoplasmic ornithine decarboxylase in developing embryos of Musca domestica.

During embryonic development of Musca domestica inactive ornithine decarboxylase protein appears in the embryos at 6 h postoviposition, increases in concentration and reaches a maximum level at 9 h postoviposition. The inactive enzyme is associated with the plasma membrane and appears to be the precursor for active ornithine decarboxylase, which is associated with the cytosolic fraction just prior to hatching. Both ornithine decarboxylase protein and enzymatic activity disappear during the early larval stage of this insect.

Salt stimulation of the activation of latent protein phosphatase, Fc.M, by Mn++ and by Mn/trypsin.

The activation of porcine heart latent protein phosphatase (Fc.M) by pretreatment with Mn++ followed by trypsin (Mn/trypsin) can be stimulated 2.5-fold by including NaCl or KCl in the activation mixtures. The salts also stimulated the activation of the enzyme by Mn++ to the same level as that obtained by Mn/trypsin pretreatment in the absence of salt. The presence of salt in both the Mn++ and Mn/trypsin activations decreased the Mn++ requirement 10-fold in each case. Treatment of latent Fc.M by Mn/trypsin in the presence of 0.2 M NaCl or KCl offers a convenient method of expressing the full potential activity of the protein phosphatase.

Identification and partial characterization of bovine heart cytosolic phosphorylase phosphatases.

The properties of phosphatases in bovine heart cytosol were studied. Two isozymic forms of protein phosphatase H (H-1 and H-2) were resolved by chromatography on DEAE-Sephacel. The two isoenzymes had identical physical properties (Mr 260,000, 7.9 S). Treatment with 80% ethanol activated both isozymes and converted H-1 to a Mr 35,500 form and H-2 to Mr 67,000 and Mr 35,500 forms. Both H-1 and H-2 and their lower Mr activated forms had essentially identical Km values for phosphorylase a. The heart cytosol also contained a latent phosphatase (Fc) which could be activated by preincubation with either ATP X Mg and an activating factor (FA), or by Mn/trypsin treatment. The latter procedure converted the latent Fc (Mr 200,000) to a Mn2+-independent Mr 34,500 form. Both activated forms of Fc had similar Km values which were fourfold lower than the affinity of the protein phosphatase H forms for the phosphorylase a substrate.

Purification of bovine heart glycogen synthase.

Glycogen synthase was purified to apparent homogeneity from bovine heart muscle by a procedure involving precipitation of the enzyme in the presence of added glycogen by polyethylene glycol, chromatography on DEAE-Sephacel, and high-speed centrifugation through a sucrose-containing buffer. The enzyme was maintained in the presence of glycogen during the isolation procedure. Glycogen synthase I and D preparations were obtained having specific activities of 21-25 and 30-35 units/mg protein at pH 7.8 and 30 degrees C and having activity ratios of 0.5-0.6 and 0.05-0.10, respectively, when assayed in the absence and in the presence of glucose 6-P.