PINP: a serum biomarker of bone formation in the rat.

Serum PINP has emerged as a reliable marker of bone turnover in humans and is routinely used to monitor bone formation. However, the effects of PTH (1-34) on bone turnover have not been evaluated following short-term treatment. We present data demonstrating that PINP is an early serum biomarker in the rat for assessing bone anabolic activity in response to treatment with PTH (1-38). Rat serum PINP levels were found to increase following as few as 6 days of treatment with PTH (1-38) and these increases paralleled expression of genes associated with bone formation, as well as, later increases in BMD. Additionally, PINP levels were unaffected by treatment with an antiresorptive bisphosphonate. PINP may be used to detect PTH-induced early bone formation in the rat and may be more generally applicable for preclinical testing of potential bone anabolic drugs.

Effects of parathyroid hormone on Wnt signaling pathway in bone.

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.

Parathyroid hormone-(1-34) enhances aggrecan synthesis via an insulin-like growth factor-I pathway.

During endochondral bone formation, the growth plate chondrocytes proliferate, become hypertrophic, lose the cartilage phenotype, undergo mineralization, and provide a scaffold upon which subsequent longitudinal bone growth occurs. Parathyroid hormone (PTH), a calcium-regulating hormone, and parathyroid hormone-related peptide (PTHrP), which shares several properties with PTH, have profound effects on skeletal growth and new bone formation. In order to define further the mechanism by which PTH/PTHrP promotes the cartilage phenotype, chondrocytes isolated from the rib cages of developing rat embryos were evaluated for the biosynthesis of aggrecan. Cells treated with PTH-(1-34) for a 4-h period followed by a 20-h recovery period showed a significant increase in cartilage proteoglycan (aggrecan) synthesis in a dose-dependent manner. Only N-terminally intact PTH and PTHrP were effective in stimulating aggrecan synthesis. Addition of a neutralizing antibody to insulin-like growth factor-I (IGF-I) during PTH treatment resulted in the inhibition of PTH-stimulated aggrecan synthesis, whereas the addition of a neutralizing antibody to insulin-like growth factor-binding protein-2 (IGFBP-2) resulted in an increase in synthesis in both the control and PTH-treated cells. In addition, PTH treatment resulted in an increase in the mRNA for aggrecan, a reduction in IGFBP-3 mRNA, and no discernible changes in IGF-I mRNA levels, which was complemented by quantitative changes in IGFBP-3 and free IGF-I levels. The reciprocal relationship in the expression of aggrecan and IGFBP was further confirmed in chondrocytes from various gestational stages during normal development. Collectively, our results indicate that the effect of PTH may be mediated at least in part through the regulation of the IGF/IGFBP axis, by a decrease in the level of IGFBP-3, and an increase in free IGF-I levels. It is likely that the local increase in IGF-I may lead to an increase in cartilage type proteoglycan synthesis and maintenance of the cartilage phenotype. The consequence of the prolonged maintenance may be to halt mineralization while a new scaffolding is created.

Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.

Development of a scintillation proximity assay for high-throughput measurement of intact parathyroid hormone.

A simple, high-throughput scintillation proximity assay (SPA) for parathyroid hormone (1-84) (PTH) has been developed. Fifteen commercially available N-terminal and six C-terminal anti-PTH antibodies were evaluated for detection of human PTH(1-84). Two C-terminal antibodies (CR1073M and 10-P55) gave the most consistent results. Using one of these antibodies (10-P55), an assay was developed with a sensitivity of 4 pg/ml for human and rat PTH(1-84). Porcine PTH(1-84) was not detectable. The intra-assay and inter-assay coefficients of variation for a 467 pg/ml sample were 6. 1 and 6.5%, respectively, and for a 21 pg/ml sample, 6.2 and 4.4%. Human PTH(1-34), while not detected in the assay, interfered with the detection of PTH(1-84). Smaller fragments [for example, human PTH(3-34)] and a C-terminal PTH fragment [PTH(53-84)] did not interfere in the assay. The procedure gave 106-110% recovery of human PTH(1-84) spiked into samples. Immunoreactive PTH concentrations in serum of rats administered EGTA were determined by SPA and by a commercially available PTH immunoassay. There was a good correlation between the two assays with significant increases in serum immunoreactive PTH concentrations at 15 and 30 min after EGTA injection and a rapid decrease to baseline values by 60 min. The SPA gives a high-throughput method for simply and accurately determining PTH(1-84) concentrations in serum.

Evaluation of the major metabolites of raloxifene as modulators of tissue selectivity.

Raloxifene (LY139481 HCl) is a selective estrogen receptor modulator (SERM) which blocks the effects of estrogen on some tissues, such as the breast and uterus, while mimicking estrogen in other tissues, such as bone. To study the origins of this unique pharmacology, we have prepared the major metabolites of raloxifene as chemical probes for examining the estrogen receptor function in vitro and in vivo. In human breast cancer cell (MCF-7) related assays, these glucuronide conjugates show little affinity for the estrogen receptor and are more than two orders of magnitude less potent at inhibiting cell proliferation than raloxifene. In non-traditional estrogen target tissue, such as bone, these metabolites are less effective than the parent at inhibiting cytokine-stimulated bone resorbing activity in rat osteoclasts or producing transforming growth factor beta-3 (TGF-beta3). In animal models, tissue distribution studies with radiolabelled metabolite indicate that conversion to raloxifene occurs readily in a variety of tissues including the liver, lung, spleen, kidney, bone and uterus. Differential conversion of metabolite in target organs, such as bone and the uterus, is not observed indicating that the origin of raloxifene's pharmacology does not result from tissue-selective deconjugation of metabolite to parent.

Structure and functional expression of a complementary DNA for porcine parathyroid hormone/parathyroid hormone-related peptide receptor.

A complementary DNA (cDNA) encoding the receptor for porcine parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) was isolated from a porcine kidney cDNA library. The porcine PTH/PTHrP receptor is a 585 amino acid protein containing seven putative membrane-spanning domains. The porcine PTH/PTHrP receptor has amino acid identity of 95.6%, 80.4%, and 88.7% with human, opossum, and rat PTH/PTHrP receptors, respectively and 53.4% identity to the recently cloned human PTH2 receptor. The receptor cDNA was subsequently cloned into a mammalian cell expression vector (pRC/CMV) which contains a human cytomegalovirus promoter. A human kidney cell line (293), stably transfected with this vector, expressed the receptor at a high level and, when challenged with human PTH(1-34), increased cytoplasmic cAMP and inositol triphosphate production. Radioligand binding studies revealed that the receptor bound both human PTH(1-34), and PTHrP(1-36). Scatchard analyses of three clones showed that the cells harbor a single class of high affinity receptor (Kd = 1-4 nM for human PTH(1-34)) but had varying receptor numbers (10(5)-10(6) receptors/cell). In contrast to PTH(1-34), the [Arg2]PTH(1-34) analog bound to the porcine PTH/PTHrP receptor with low affinity and was a weak agonist for cAMP stimulation with the cloned receptor. These response characteristics differentiate the porcine receptor from the previously cloned rat and opossum PTH/PTHrP receptors.

Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate.

Bone loss associated with postmenopausal osteoporosis can be reduced by treatment with antiresorptive agents such as estrogen or bisphosphonates. Whereas bisphosphonates primarily affect bone loss, estrogens have an advantage of also lowering serum cholesterol levels, although they have a detrimental effect in the uterus. Recently, raloxifene HCl, a selective estrogen receptor modulator (SERM), has been shown to decrease both bone loss and cholesterol levels without the negative uterine effects. These antiresorptive agents reduce bone turnover, which can be evaluated by measuring bone turnover markers. To compare the effects of estrogen, two SERMs (raloxifene HCl and tamoxifen), and alendronate, a bisphosphonate that inhibits bone loss by an estrogen-independent pathway, on metabolic bone markers and cholesterol levels, rats were ovariectomized 2 weeks prior to 3 weeks of daily oral treatment with raloxifene HCl (3 mg/kg), ethynyl estradiol (0.1 mg/kg), tamoxifen (3 mg/kg), or alendronate (3 mg/kg). Raloxifene HCl, tamoxifen, and ethynyl estradiol reduced serum cholesterol to levels below control values within 4 days after initiation of treatment, whereas alendronate had no effect. After 3 weeks of treatment, serum cholesterol values in ethynyl estradiol treated animals, although still below the control value, had risen 6.4-fold; raloxifene HCl and tamoxifen values rose by only 1.4-1.5-fold. Therefore, compared with estrogen, SERMs may have a longer-term suppressive effect on serum cholesterol. At 4 days of treatment, ovariectomized rats had a 1.4-fold increase in serum osteocalcin level compared with controls. Ethynyl estradiol lowered this level within 1 week of treatment by 18%, with a more pronounced reduction of 34% at 3 weeks. In contrast, raloxifene HCl, tamoxifen, or alendronate had very little effect after the first week (6% to 13% reduction), although there was an 18% to 25% reduction by 3 weeks. Urinary pyridinoline levels, elevated 1.4-fold in the ovariectomized rat compared with controls 2 weeks after surgery, were reduced to control values after 2 weeks of treatment with raloxifene HCl, ethynyl estradiol, tamoxifen, or alendronate. These data support the concept that estrogen, raloxifene HCl, tamoxifen, and alendronate inhibit bone loss in the ovariectomized animal by reducing bone resorption. The results also indicate that for treatment of postmenopausal osteoporosis, raloxifene HCl may have an advantage over the other antiresorptives studied in having both non-uterotrophic and hypocholesterolemic effects in addition to its ability to inhibit bone resorption.

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR studies of the active site of isopenicillin N synthase, a non-heme iron(II) enzyme.

The active site structure of isopenicillin N synthase (IPNS) has been previously studied by the use of Mössbauer, EPR, electronic absorption, and NMR spectroscopies [Chen, V.J., Frolik, C.A., Orville, A.M., Harpel, M.R., Lipscomb, J.D., Surerus, K.K., & Münck, E. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C.A., & Chen, V.J. (1990) Inorg. Chem. 26, 1111-1112]. These studies have revealed three coordinated His residues along with three sites for substrate [delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine, ACV], NO, and water binding on the active Fe(II) of IPNS. We report here NMR studies of Fe(II)IPNS and its Co(II)-substituted derivative [Co(II)IPNS]. By the use of NOE techniques on the Co(II)IPNS-ACV complex, we have recognized a -CH2-CH less than spin system at 14.6, 24.3, and 38.6 ppm that is assigned to the alpha and beta protons of a coordinated Asp residue. Corresponding solvent nonexchangeable features are found near 40 ppm in Fe(II)IPNS and the Fe(II)IPNS-ACV complex, but the peaks are too broad for NOE effects to be observed. The binding of NO to the Fe(II) center results in a significant change in the configuration of the metal site: (a) The C beta H2 resonances due to the coordinated Asp residue disappear. The loss of the signal may indicate a change of the carboxylate configuration from syn-like to anti-like or, less likely, its displacement by NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of insulin-like growth factors to Tera-2 human embryonal carcinoma cells during differentiation.

Differentiation of Tera-2 human embryonal carcinoma cells by exposure to 2.1 mM alpha-difluoromethylornithine resulted in changes in morphology, a decrease in growth rate, and changes in the expression of SSEA-1 differentiation antigen. While the binding of 125I-insulin-like growth factor I (IGF-I) remained relatively constant during differentiation, binding of 125I-IGF-II increased 2-3-fold. Further, the binding of IGF-II was 87 times greater than IGF-I in both undifferentiated and differentiated cells. Undifferentiated Tera-2 cells exhibited a single class of binding sites for both IGF-I (KD = 1.2 nM, 7.0 x 10(3) sites/cell) and IGF-II (KD = 8.3 nM, 3.4 x 10(5) sites/cell). Following differentiation, IGF-I continued to bind to a single class of binding sites (KD 1.0 nM, 6.7 x 10(3) sites/cell) whereas IGF-II bound to both high-affinity sites (KDH 0.3 nM, 2.2 x 10(5) sites/cell) and low-affinity sites (KDL 15.1 nM, 1.6 x 10(7) sites/cell). The binding of iodinated IGF-II was blocked by unlabeled IGF-II but not IGF-I. In contrast, 125I-IGF-I binding was prevented by either IGF-I or IGF-II. Affinity cross-linking experiments demonstrated the presence of both type I and type II IGF receptors along with a number of IGF binding proteins. IGF-I failed to stimulate the incorporation of [3H]thymidine in both undifferentiated and differentiated cells. Although IGF-II caused a significant increase in [3H]thymidine incorporation in both undifferentiated and differentiated Tera-2 cells, the magnitude of the response and the sensitivity of the cells to IGF-II stimulation was diminished following differentiation. The observed changes in IGF-II binding, which occur in conjunction with cellular differentiation, may be an important feature of the expression of the differentiated phenotype by human germ cell tumors.

Binding of insulin-like growth factors to cultured rat calvarial cells with differing biologic responses.

The insulin-like growth factors (IGFs) are considered important regulators of bone metabolism affecting a number of biologic responses in vitro. Primary fetal rat calvarial cells (PRC) and a cloned adult rat calvarial cell line (C3) both exhibit a concentration-dependent IGF stimulation of [3H]thymidine incorporation into DNA, but the C3 cells show a greater sensitivity and magnitude of response. IGF-I and IGF-II were nearly equipotent in PRC cultures, but IGF-I was more than twice as active as IGF-II in the C3 cultures. This effect of the IGFs on DNA synthesis in two bone cell cultures with different culture histories has been correlated with receptor and binding protein profiles. Specific high-affinity IGF binding sites were found in both cell types. In general, the sites present on PRC cells showed a preference for binding IGF-II over IGF-I, but C3 cells displayed two types of relatively specific binding sites. In both cell types [125I]IGF-I bound primarily to a protein with IGF type I receptor characteristics. However, in PRC cells, [125I]IGF-II cross-linked specifically with proteins that had IGF type II receptor characteristics plus several sites unique to these cells; in C3 cells, [125I]IGF-II bound to a 139 kD protein that could be displaced by either IGF-I or IGF-II. Finally, IGF-II-specific 85 and 67 kD proteins were common to both cell types. From these studies, it is apparent that the IGFs bind to a variety of high-affinity binding sites in bone cells and that these sites differ between a highly responsive and a less responsive bone cell population.

The functional role of cysteines in isopenicillin N synthase. Correlation of cysteine reactivities toward sulfhydryl reagents with kinetic properties of cysteine mutants.

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium contains 2 cysteine residues in positions 106 and 255 which are invariant in all IPNS sequences reported to date (Miller, J.R., and Ingolia, T.D. (1989) Mol. Microbiol. 3, 689-695). Although these residues have been postulated to play a role in catalysis (Samson, S.M., Chapman, J.L., Belagaje, R., Queener, S., and Ingolia, T.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5705-5709) as well as enzyme inactivation (Perry, D., Abraham, E.P., and Baldwin, J.E. (1988) Biochem. J. 255, 345-351) little information exists regarding their oxidation state and reactivity. In this paper, the functions of these cysteines have been addressed by chemical modification techniques in combination with site-directed mutagenesis. In the intact wild type protein, both cysteines are inert toward 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetic acid. However, Cys-106, but not Cys-255, can be slowly modified by N-ethylmaleimide, and its modification is partially blocked by the presence of a substrate analog inhibitor. Complete modification of both cysteines by sulfhydryl reagents requires unfolding of the protein but not the presence of a disulfide reducing agent. The thiol content of IPNS is shown to be the same before and after exposing the enzyme to substrate even though during catalysis the enzyme is rapidly inactivated. The impact on catalysis due to alteration of the cysteines has been assessed using three site-specific mutants: Cys-106----Ser, Cys-255----Ser, and Cys-106,255----Ser. These mutant proteins have been purified as apoenzymes with the nature of the mutation verified by peptide mapping. The stoichiometry of metal required for activity remains as one equivalent of Fe2+/mol of enzyme in the mutants as in wild type IPNS. Compared with wild type, Cys-255----Ser shows a reduction in Vmax by 33%, and an increase in Km by 1.4-fold, while Cys-106----Ser and Cys-106,255----Ser, which have identical kinetic properties, exhibit a decrease in Vmax by 63% but an elevation of Km by 14-fold. The data presented demonstrate that 1) both cysteines are free thiols; 2) Cys-106 is more exposed than Cys-255; 3) substrate-induced inactivation is not caused by cysteine modification; 4) neither cysteine is absolutely essential for bond making or breaking events during catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Physiological and pharmacological regulation of biological calcification.

Biological calcification is a highly regulated process which occurs in diverse species of microorganisms, plants, and animals. Calcification provides tissues with structural rigidity to function in support and protection, supplies the organism with a reservoir for physiologically important ions, and also serves in a variety of specialized functions. In the vertebrate skeleton, hydroxyapatite crystals are laid down on a backbone of type I collagen, with the process being controlled by a wide range of noncollagenous proteins present in the local surroundings. In bone, cells of the osteoblast lineage are responsible for the synthesis of the bone matrix and many of these regulatory proteins. Osteoclasts, on the other hand, are continually resorbing bone to both produce changes in bone shape and maintain skeletal integrity, and to establish the ionic environment needed by the organism. The proliferation, differentiation, and activity of these cells is regulated by a number of growth factors and hormones. While much has already been discovered over the past few years about the involvement of various regulators in the process of mineralization, the identification and functional characterization of these factors remains an area of intense investigation. As with any complex, biological system that is in a finely tuned equilibrium under normal conditions, problems can occur. An imbalance in the processes of formation and resorption can lead to calcification disorders, and the resultant diseases of the skeletal system have a major impact on human health. A number of pharmacological agents have been, and are being, investigated for their therapeutic potential to correct these defects.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate.

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mössbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.

Isolation and characterization of insulin-like growth factor-II from human bone.

Human bone was sequentially extracted with 4 M guanidine hydrochloride to remove nonmineralized tissue components, 0.5 M EDTA to dissolve the mineral phase, 4 M guanidine hydrochloride to remove matrix associated proteins and finally a combination of 4 M guanidine hydrochloride and 0.5 M EDTA to remove residual proteins. The extracts were examined for the presence of factors that were able to stimulate the incorporation of [3H] thymidine into DNA and [14C] leucine into protein in a cloned rat bone cell culture system. The majority of the bioactivity was found in the first guanidine hydrochloride extract (59 +/- 12%) while the second guandine hydrochloride extract contained 27 +/- 8%. In addition to several known growth factors already reported to be present in bone (transforming growth factor-beta and insulin-like growth factor-I) insulin-like growth factor-II was identified by its chromatographic, electrophoretic and immunological properties as well as by N-terminal sequence data. The insulin-like growth factor-II levels (802 +/- 112 micrograms/kg wet weight bone) were 10 fold higher than that found for insulin-like growth factor-I (84 +/- 23 micrograms/kg wet weight).

Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha.

Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate-polyacrylamide gels, the apparent molecular weight of these intracellular TGF-alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF-beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate-polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta.

Characterization of a membrane receptor for transforming growth factor-beta in normal rat kidney fibroblasts.

A procedure has been developed for the iodination of human transforming growth factor-beta (TGF-beta) with full retention of biological activity. Using the iodinated peptide, saturable receptors have been found for TGF-beta on normal rat kidney fibroblasts, a cell line that will grow in soft agar in the presence of TGFs but not in their absence. Scatchard analysis of the binding data showed a high affinity binding site (dissociation constant equal to 25 to 30 pM with approximately 17,000 receptors per cell). The receptor was specific for TGF-beta with epidermal growth factor, insulin, insulin-like growth factors I and II, platelet-derived growth factor, and TGF-alpha being unable to compete for the binding of 125I-TGF-beta to the receptor. The binding of TGF-beta was a time- and temperature-dependent process. At 37 degrees C, maximal binding was attained within 45 to 60 min after addition of 125I-TGF-beta followed by a rapid decline in cell-associated radioactivity due to degradation of the 125I-TGF-beta. As demonstrated using ammonium acetate, a compound known to inhibit lysosomal enzymes, this degradation was most likely due to the action of proteolytic enzymes found in the lysosome. At 0 degrees C, binding reached a plateau within 2 to 3 h and maintained this level with no apparent drop during the 4-h incubation period. The receptor could be down-regulated by TGF-beta, but not by epidermal growth factor, to approximately 50% of the level initially observed.