Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.     

Mycoplasma growth factors in bovine serum fraction.

Mycoplasma growth factors in bovine serum fraction were separated by Sephadex G150 column chromatography and density ultracentrifugation. The major growth factor of bovine serum fraction eluted from the Sephadex column in the void volume. Its growth-supporting activity was greatly enhanced by the presence of bovine serum albumin in the mycoplasma culture media. Other investigators had previously identified the major growth factor in serum as an alpha-lipoprotein. Although density ultracentrifugation revealed the presence of traces of a high-density lipoprotein in bovine serum fraction, another, less dense component, isolated by ultracentrifugation (component 3) and containing cholesterol, cholesteryl esters, free fatty acids, triglycerides, and protein, but no lipoprotein, exhibited considerably more growth-supporting activity than did the high-density lipoprotein, thus indicating that at least two mycoplasma species do not require intact serum lipoprotein for growth. Both the high-density lipoprotein and component 3 exhibited maximum activity only in the presence of bovine serum albumin. A chloroform extract containing component 3 lipids combined with bovine serum albumin to form an effective, partially defined, less complex substitute for serum in mycoplasma culture media.     

Insulin-like growth factor I (IGF I) induces cortisol production in bovine adrenocortical cells in primary culture

The effects of a physiological dose of IGF I (40 ng/ml approximately 5 x 10(-9) M) on steroidogenesis were studied in bovine adrenal fasciculata cells cultured in serum-free McCoy's medium. They were compared with those of a single dose of ACTH (0.25 ng/ml approximately 10(-10) M) at approximately the concentration inducing half-maximal stimulation. With IGF I, steroidogenesis commenced after 48 h culture and progressively increased throughout the 96-h test period. Expressed as stimulated level/control level ratios, glucocorticoid (cortisol + corticosterone) responses to IGF I after 4 days' culture (2.41 +/- 0.20 (SEM) n = 9) were similar to those obtained with ACTH (2.59 +/- 0.18, n = 9). A combination of the two peptides had a synergistic effect (5.95 +/- 0.79, n = 5). The cortisol/corticosterone ratio increased in the presence of IGF I from 1 +/- 0.19 to 1.76 +/- 0.45 (n = 7, P less than 0.02), although less so than in the presence of ACTH (5.50 +/- 0.98). Moreover, cortisol production was accompanied by androstenedione production (2.36 ng/10(6) cells, n = 3) similar to that induced by ACTH (2.10 ng/10(6) cells, n = 3). These findings together suggest stimulation of 17 alpha-hydroxylase activity. Cell multiplication was unaffected by IGF I. [3H]Thymidine incorporation into DNA reached only 193% +/- 17 (SEM) (n = 4) of control levels, whereas with ACTH it dropped to 60% +/- 5. Our findings show that IGF I alone has no mitogenic effect on adrenocortical cells in vitro, but that it is capable of inducing differentiated steroidogenesis.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

Insulin-like growth factors in fetal macrosomia with and without maternal diabetes

Insulin-like growth factors (IGFs/somatomedins) have been implicated as regulators of fetal growth. This study investigates whether IGFs are related to macrosomia in infants of normal or insulin-dependent diabetic mothers. Cord concentrations of IGF-I (radioimmunoassay), total IGF (radioreceptor assay) and IGF binding protein (radiobinding assay) were measured in 15 term infants of diabetic mothers (IDM) and 29 term infants of nondiabetic mothers. In infants of control mothers cord IGF and total IGF levels were significantly higher in large-for-gestational-age than appropriate-for-gestational-age infants; but this relationship was lost in IDM, in whom IGF-I concentrations were similar to control infants. IGF binding protein levels were not significantly different in any of these groups. The absence of elevated IGF levels in macrosomic IDM indicates that the pathologic process does not involve a simple increase in these growth factors.     

Insulin-like growth factors as mediators of functional plasticity in the adult brain.

Insulin-like growth factors (IGFs) are present in the brain throughout life. While their role as modulators of brain growth and differentiation during development is becoming apparent, their possible involvement in adult brain function is less known. Nevertheless, accumulating evidence indicates a role for IGFs in brain plasticity processes. Specifically, IGFs modulate synaptic efficacy by regulating synapse formation, neurotransmitter release and neuronal excitability. IGFs also provide constant trophic support to target cells in the brain and in this way maintain appropriate neuronal function. Pathological dearrangement of this trophic input may lead to brain disease. Molecular targets of the IGFs in the adult brain may include pre- and post-synaptic proteins involved in synaptic contacts, membrane channels, neurite-guiding molecules, extracellular matrix components and glial-derived intercellular messengers. Future studies on the role of IGFs in the adult brain may help unravel the relationship between neuronal plasticity and brain disease.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro

The role(s) of one family of polypeptide growth factors in a developing organ system was examined. Renal anlagen (metanephroi) were surgically removed from 13-d-old rat embryos and grown in organ culture for up to 6 d. Over this period of time when placed in serum-free defined media, the metanephroi increased in size and morphologic complexity. Messenger RNAs for both insulin-like growth factors (IGFs), IGF I and IGF II, were present in the metanephroi. Immunoreactive IGF I and IGF II were produced by the renal anlagen and released into culture media. Levels were relatively constant during the 6 d in culture and averaged 3.5 X 10(-9) M IGF I and 8.3 X 10(-9) M IGF II in media removed from metanephroi after contact for 24 h. IGF binding protein activity was not detected in culture media. Growth and development of metanephroi in vitro was prevented by the addition of anti-IGF I or anti-IGF II antibodies to organ cultures. IGF II produced by metanephroi was active in an IGF II biological assay system and addition of anti-IGF II receptor antibodies to organ cultures prevented growth and development, consistent with the action of IGF II in metanephroi being mediated via the IGF II receptor. The data demonstrate production of both IGF I and IGF II by developing rat metanephroi in organ culture. Each of these peptides is necessary for growth and development of the renal anlage to take place in vitro. Our findings suggest that both IGF I and IGF II are produced within the developing metanephros in vivo and promote renal organogenesis.     

Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G4 enzyme

Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.     

Insulin-like growth factor I levels and the diagnosis of adult growth hormone deficiency

The current guidelines state that, within the appropriate clinical context, the diagnosis of adult growth hormone (GH) deficiency must be made biochemically using provocative tests. Measurement of insulin-like growth factor I (IGF-I) and binding protein 3 (IGFBP-3) levels cannot always distinguish between healthy and GH-deficient individuals. In particular, IGFBP-3 as a marker of GH status is clearly less sensitive than IGF-I and there is general agreement that its measurement does not provide useful diagnostic information. However, the diagnostic value of measuring IGF-I levels has been revisited recently. It has been confirmed that normal IGF-I levels do not rule out severe GH deficiency (GHD) in adults, in whom the diagnosis has therefore to be based on the demonstration of severe impairment of the peak GH response to provocative tests. It has also been emphasized that very low IGF-I levels in patients with high suspicion of GHD could be considered to be definite evidence for severe GHD. This assumption particularly applies to patients with childhood-onset, severe GHD or with multiple hypopituitary deficiencies acquired in adulthood. In addition, the use of IGF-I levels to monitor the efficacy and adequacy of recombinant human GH replacement remains widely accepted.     

Similarities in structure and function of bovine serum erythrotropin and human insulin-like growth factor II: two fetal erythroid cell stimulating factors

Serum erythrotropin (ET) was isolated from fetal bovine serum. Partial sequence analysis of the N-terminal portion of the peptide indicated that the first 20 amino acids were practically identical to those found in human insulin-like growth factor II (IGF II). The effect of IGF II on [3H] thymidine incorporation in cell cultures of fetal bovine liver was similar to the effect of ET. Both factors acted synergistically with erythropoietin but not with platelet derived growth factor. The stimulation of thymidine incorporation by ET and IGF II on cell cultures of fetal liver erythroid cells was at least 15 times higher than their effects on cultures of fetal calf intestine, lung and kidney cells.     

Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis.

beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through beta-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgalactosaminyltransferase and beta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin-synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both beta-N-acetylgalactosaminyltransferase II and beta-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.     

Bovine-associated mucoprotein: I. Distribution among adult and fetal bovine tissues and body fluids

Bovine-associated mucoprotein (BAMP), solubilized with water from the delipidated membranes of bovine milk fat globules, is not restricted to fat globules or to the alveolar epithelial cells from which they are formed. BAMP also has a widespread distribution on other bovine glandular epithelial cells and on undifferentiated cells in lymphoid germinal centers and in several fetal tissues. Free BAMP is present in bovine colostrum, milk, other secretory fluids, and in fetal serum but is absent from adult and colostrum-deprived calf sera. In bronchoalveolar fluids, BAMP is preferentially found in the mucus-rich fraction. BAMP is antigenically distinct from all adult serum proteins, free secretory component, beta 2-microglobulin, lactoferrin, alpha-lactalbumin, beta-lactoglobulin, and five different caseins. BAMP as a free protein constitutes one-sixth of the total amount of BAMP present in milk. The BAMP-related component of fetal serum lacks antigenic determinants present on the BAMP of milk as demonstrated by immunoprecipitation and partial blocking of immunofluorescence. The fetal component is not fetuin or alpha 1-fetoprotein. These data suggest that BAMP may be useful in studies of the membranes of proliferating or differentiating epithelial cells.