Characterization of a high-molecular-weight form of epidermal growth factor in an extract of human urine.

We purified from a side fraction of the commercial preparation of urokinase from large volumes of human urine a high-molecular-weight (HMW) form of human epidermal growth factor (hEGF). Sequence analysis of the amino terminus of the intact molecule and of two tryptic fragments and carboxypeptidase Y analysis revealed the molecule to correspond to residues 828-1023 of the hEGF precursor predicted by the nucleotide sequence of human renal hEGF mRNA, with hEGF forming its carboxyl terminus. HMW hEGF bound poorly to concanavalin A-agarose, quite avidly to wheat germ lectin-agarose, and completely to phenyl boronate-agarose, suggesting that it was O-glycosylated. Sephacryl S-200 chromatography of freshly-voided urine revealed mostly hEGF, with smaller amounts of a much higher molecular weight hEGF, but little material that was the size of the HMW hEGF we characterized. The large fragment we characterized presumably is cleaved from the larger form by enzyme(s) present in urine during the collection, storage, and processing of urine. We have confirmed that hEGF is synthesized as a large precursor molecule, as predicted by the nucleotide sequence of hEGF mRNA.

Specific high-affinity binding protein for human corticotropin-releasing hormone in normal human plasma.

A protein of Mr 25-40 kilodaltons in normal human plasma binds human corticotropin-releasing hormone (hCRH), but not ovine CRH. Binding requires both N- and C-terminal hCRH sequences and has a Kd of 2 X 10(-10) M and a binding site concentration of 1.4 pmoles per ml of plasma. Its binding is not affected by heating to 56 degrees C for 1 h, but is abolished by exposure to 6 M guanidine-HCl, 10 mM dithiothreitol. Binding proceeds rapidly at 37 degrees C and is 75% complete within 4 min. Neither rat nor sheep plasma appears to contain a CRH-binding protein. CRH-binding protein may explain the brief biological action of hCRH as compared to ovine CRH in man and why high concentrations of plasma immunoreactive hCRH in women during third-trimester pregnancy do not cause increased ACTH secretion.

Human brain tumor-associated urinary high molecular weight transforming growth factor: a high molecular weight form of epidermal growth factor.

Urinary protein obtained from a patient with a highly malignant brain tumor (astrocytoma, grade IV) was adsorbed to trimethylsilyl controlled-pore glass beads and selectively eluted with acetonitrile to yield a high molecular weight (HMW) human transforming growth factor (hTGF). This HMW hTGF promoted clonogenic cell growth in soft agar and competed for membrane receptors with mouse epidermal growth factor. After surgical resection of the tumor, no HMW hTGF was found in urine. HMW hTGF generated a human EGF (hEGF) radioimmunoassay competitive binding curve similar to that of hEGF and parallel to that of a highly purified HMW form of hEGF previously reported to be present in trace concentrations in normal human urine. Both hEGF and HMW hEGF were clonogenic in soft agar, and their clonogenic activity as well as that of HMW hTGF was inhibited by anti-hEGF serum. Both HMW hTGF and HMW hEGF had 20 to 25% of the radioreceptor binding activity of hEGF. HMW hTGF purified from the pooled urine of several patients with malignant astrocytomas and HMW hEGF purified from normal control urine comigrated at Mr 33,000. Thus, HMW hTGF was indistinguishable from HMW hEGF in terms of apparent molecular size, epidermal growth factor receptor binding activity, epidermal growth factor immunoreactivity, and clonogenic activity. Urinary HMW hEGF/hTGF may be of tumor cell origin or may represent a response of normal host tissues to the tumor or its products.

Purification and characterization of epidermal growth factor (beta-urogastrone) and epidermal growth factor fragments from large volumes of human urine.

We purified human epidermal growth factor (hEGF) and hEGF fragments from a benzoic acid precipitate of the materials not adsorbed to silica gel in 330 liters of human urine by a relatively brief, simple, and efficient method employing sequential batch absorption to and stepwise elution from CM-cellulose and DEAE-cellulose, Bio-Gel P-10 chromatography in 50 mM HCl, and three reverse-phase HPLC steps for final resolution and purification of hEGF components. Recovery of hEGF was 29%. Eight apparently homogeneous hEGF components were recovered, each of which had similar activities in a homologous hEGF radioimmunoassay and an EGF radioreceptor assay using human placental membranes. Amino acid composition analysis indicated that there were four pairs of components that represented intact 53-amino acid hEGF, hEGF-(1-52), hEGF-(1-51), and hEGF-(1-50); intact hEGF accounted for one-third of the total materials recovered. Automated Edman degradation of each component for at least 10 cycles revealed a single amino acid sequence identical to that proposed for human beta-urogastrone. Similar immunoreactive hEGF components were observed in similar proportions in freshly voided urine, indicating that they were not artifacts of the purification process. Thus, multiple forms of fully biologically active hEGF (i.e., beta-urogastrone) can be relatively easily and efficiently purified from large volumes of human urine.

Epidermal growth factor in human milk: daily production and diurnal variation during early lactation in mothers delivering at term and at premature gestation.

Epidermal growth factor is a polypeptide that stimulates proliferation and differentiation of a variety of cell types, including the developing intestinal epithelium; it is the agent in human milk that induces mitosis in human fibroblast culture. We systematically evaluated the EGF content of milk from 20 women delivering prematurely and from 11 women delivering at term. In preterm mothers, the concentration of EGF was 70 +/- 5 ng/ml (mean +/- SEM), with no significant change during seven weeks of lactation. EGF concentration in milk of term mothers was 68 +/- 19 ng/ml (mean +/- SEM). No diurnal variation in the concentration was found. Total EGF content was closely correlated with the volume of milk expressed, suggesting a passive transport from the circulation. These observations confirm that a substantial amount of EGF is present in human milk and that EGF concentrations are not affected by duration of gestation, time of day, or duration of lactation.

NH2-terminal amino acid sequence and peptide mapping of purified human beta-lipotropin: comparison with previously proposed sequences.

Beta-Lipotropin was purified from human pituitary glands to a purity of greater than 90%. The amino acid compositions of beta-lipotropin and its three cyanogen bromide cleavage peptide fragments were in agreement with the structure proposed by Li and Chung [Li, C.H. & Chung, D. (1981) Int. J. Pept. Protein Res. 17, 131-142]. However, the amino acid sequence of its NH2-terminal 46 amino acid residues established here differs both from the sequence derived from the direct sequence analysis of the peptide reported by Li and Chung and from that predicted on the basis of the nucleotide sequence of the human pro-opiolipomelanocortin gene proposed by Chang et al. [Chang, A.C.Y., Cochet, M. & Cohen, S.W. (1980) Proc. Natl. Acad. Sci. USA 77,4890-4894] but agrees with the structure recently derived by direct sequence analysis by Hsi et al. [Hsi, K.L., Seidah, N.G., Lu, C.L. & Chrétien, M. (1981) Biochem. Biophys. Res. Commun. 103, 1329-1335] and predicted on the basis of nucleotide sequence analysis by Takahashi et al. [Takahashi, H., Teranishi, Y., Nakanishi, S. & Numa, S. (1981) FEBS Lett. 135, 97-102]. These discrepancies, found from residues 9 to 25 of beta-lipotropin, could result from pro-opiolipomelanocortin gene polymorphism, from the existence of multiple genes for pro-opiolipomelanocortin, or, more probably, from minor errors in nucleotide and amino acid sequence analyses.

Proopiolipomelanocortin peptides in normal pituitary, pituitary tumor, and plasma of normal and Cushing's horses.

Using RIAs for six regions within proopiolipomelanocortin (proOLMC), gel filtration, and electrophoresis, we studied pituitary peptides in a normal horse and one with Cushing's disease caused by a pars intermedia adenoma. Almost all immunoreactive (IR) ACTH (78%) was 4,500 mol wt (4.5K) ACTH in normal pars distalis, but it was almost 100% corticotropin-like intermediate lobe peptide (CLIP) in normal pars intermedia. alpha MSH and beta MSH were found mainly in pars intermedia: equal concentrations of the beta MSH precursors, beta-lipotropin (beta LPH) and gamma LPH, were found in pars distalis. Most IR-beta-endorphin (IR-beta END) was found as beta END in pars intermedia, but roughly equal concentrations of beta END and its precursor, beta LPH, were found in pars distalis. A 33K molecule containing IR-ACTH, IR-gamma 3MSH, and IR-beta END, presumed to be proOLMC, and a variety of 15-27K presumed biosynthetic intermediates were found in both normal pars distalis and pars intermedia. The pars intermedia adenoma causing Cushing's syndrome contained high IR-peptide concentrations. Several differences in precursors were noted, including the presence of three larger presumed precursors (38.5K, 47K, and 63K) that had both ACTH and beta END immunoreactivities and both deletions and additions of 15-27K intermediates. The Cushing's horse's plasma peptides reflected tumor concentrations; 4.5K ACTH was modestly elevated, but the concentrations of CLIP, alpha MSH, beta MSH, gamma LPH, and beta END were dramatically increased. About 20% of plasma IR-ACTH and 5% of IR-beta MSH and IR-beta END were found as high molecular weight forms. Normal processing of horse proOLMC appears to be similar to that in other species, but may be altered in pars intermedia tumors of horses with Cushing's disease, the plasma of which contains disproportionately increased concentrations of pars intermedia proOLMC peptides.

Effect of beta-lipotropin on aldosterone production in the isolated rat adrenal cell preparation.

Stimulation of aldosterone and corticosterone production by pituitary peptides structurally or biosynthetically related to ACTH was investigated in suspensions of isolated rat adrenal glomerulosa and fasciculata cells, respectively. Three different preparations of highly purified ovine (Li) or human (Chrétien and Orth) beta-lipotropin (beta LPH) were tested. In contrast to synthetic ACTH-(1-24), which stimulated aldosterone secretion at concentrations of 10(-12)-10(-11) M, beta LPH concentrations of 10(-8)-10(-6) M were required for significant stimulation. Stimulation of corticosterone production by beta LPH preparations generally paralleled their aldosterone-stimulating activity, and most steroidogenic activity could be accounted for by immunoreactive ACTH, as determined in two ACTH RIAs. Synthetic human beta LPH-(37-58), which contains the 47-53 heptapeptide sequence common to beta LPH and ACTH, had aldosterone- and corticosterone-stimulating activities similar to those of equimolar concentrations of beta LPH, whereas synthetic fragments of the COOH-terminal (61-91) portion of beta LPH had no steroidogenic activity. These data indicate that part of the slight steroidogenic activity of purified beta LPH preparations is due to contaminating ACTH, and part is due to the intrinsic ACTH-like activity conferred upon beta LPH by the amino acid sequence shared with ACTH. In contrast to ACTH, the concentrations of beta LPH required to stimulate adrenal steroidogenesis were 10(2)-10(5) times greater than normal plasma levels, indicating that physiological pituitary beta LPH secretion has no direct role in regulating the secretion of aldosterone.

Human gamma-lipotropin radioimmunoassay: identification of immunoreactive gamma-lipotropin in human plasma and tissue.

Disagreement exists concerning the relative contributions to total plasma immunoreactive human lipotropin (hLPH) made by h beta LPH and its amino-terminal fragment, h gamma LPH [h beta LPH-(1-58)]. Using an antiserum (R1547) which requires the free COOH-terminal Asp58 residue of h gamma LPH for full affinity and reacts only 1% as well with h beta LPH and antisera (R3 and G106) that react with both LPHs, we examined gel chromatography eluate fractions of plasma extracts from one normal subject under basal conditions and another after metyrapone administration, of plasma or plasma extracts from patients with ACTH/LPH hypersecretion of various causes, of an extract two normal pituitary glands, and an extract of an ectopic ACTH/LPH-secreting tumor. Immunoreactive h gamma LPH was always a major, frequently the predominant, and sometimes the only immunoreactive LPH observed. We also observed in plasma or tissue of two patients with ectopic ACTH/LPH syndrome a peptide whose immunoreactivity and apparent molecular size were consistent with those of octadecapeptide human beta MSH, a molecule that is not thought to exist in normal man. These studies demonstrate that h gamma LPH is a major LPH component in plasma and tissues and indicate that the h gamma LPH RIA provides a reliable estimate of the h gamma LPH concentration in plasma without prior chromatography.

Simultaneous assay of immunoreactive beta-lipotropin, gamma-lipotropin, and beta-endorphin in plasma of normal human subjects, patients with ACTH/lipotropin hypersecretory syndromes, and patients undergoing chronic hemodialysis.

We have studied the relative concentrations of the human immunoreactive (IR) peptides gamma-lipotropin (hgammaLPH, [1-58]hbetaLPH), beta-lipotropin (hbetaLPH), and beta-endorphin (hbetaEND, [61-91]hbetaLPH) using gel exclusion chromatography together with a specific radio-immunoassay (RIA) for hgammaLPH and a RIA that (because hbetaEND is the COOH-terminus of the hbetaLPH molecule) measures both hbetaEND and hbetaLPH on an equimolar basis. In normal subjects, basal plasma IR-hgammaLPH was often undetectable (<12.5 fmol/ml), but ranged up to 21 fmol/ml, and IR-hbetaEND/hbetaLPH was 10.8+/-0.7 fmol/ml; previous studies by others suggest that most of the IR-hbetaEND/hbetaLPH was probably hbetaLPH. Both IR-hgammaLPH and IR-hbetaEND/hbetaLPH were significantly elevated (P < 0.001) in patients undergoing chronic hemodialysis (101.5+/-12.7 and 23.8+/-2.0 fmol/ml, respectively). Their IR-hgammaLPH coeluted with standard hgammaLPH as a single peak, and IR-hbetaEND/hbetaLPH coeluted with hbetaLPH; no distinct peak of IR-hbetaEND was observed. In patients with ACTH/LPH hypersecretion due to Addison's disease, Nelson's syndrome, or ectopic ACTH syndrome, IR-hgammaLPH and IR-hbetaEND/hbetaLPH were both elevated, and IR-hbetaEND/hbetaLPH eluted as two peaks, one coeluting with hbetaLPH and the other with hbetaEND. The molar concentrations of all three peptides were significantly correlated with one another. The lower concentrations of endogenous IR-hbetaEND observed may be due in part to its apparent shorter plasma half-life, as estimated in an Addison's patient given a cortisol infusion. The biologic significance of these three peptides in circulating blood is still unknown. The increased levels of hbetaLPH and hgammaLPH in plasma of patients with chronic renal failure suggest that the kidney may be an important organ for their metabolism.

[Ectopic secretion of ACTH and of related peptides (LPHs, beta-endorphin, "16K"). Evidence for a common precursor]. / Sécrétion ectopique d'ACTH et de peptides apparentés (LPHs, beta-endorphine, "16K"). Mise en évidence d'un précurseur commun.

Studies in the experimental mouse pituitary tumor cell line AtT-20/D-16-v have recently shown that ACTH, the lipotropins (beta- and gamma-LPH), beta-endorphin (beta-End) and the 16-K fragment are synthesized through a common precursor molecule which is a 31,000 glycopeptide (pro-ACTH/endorphin). We have investigated whether such a biosynthetic model might exist in man. Radioimmunoassays have been developed against human ACTH, N-terminal LPH, beta-End or C-terminal beta-LPH, C-terminal gamma-LPH, and the 16-K fragment or N-terminal pro-ACTH/endorphin. These radioimmunoassays were used to examine various human samples before and after gel fractionation in ordinary or denaturing buffers. Medium DMS-79, in which human small cell carcinoma cells derived from a lung cancer were cultured, was shown to contain molecules identical to gamma-LPH, beta-LPH, beta-End and ACTH. In addition, it also contained a high molecular weight material with LPH, beta-End, and ACTH immunoreactivity. These three immunoreactivities could not be dissociated under denaturing conditions (6 M guanidine-HCl), and were all absorbed on an ACTH-purified anti-(1-24)-ACTH affinity column. Medium DMS-79 also contained high molecular weight calcitonin immunoreactivity that was not absorbed on the (1-24)-ACTH affinity column and therefore was not part of the pro-ACTH/endorphin molecule. Extracts from two pheochromocytomas responsible for the ectopic ACTH syndrome were found to contain, in addition to ACTH, large amounts of gamma-LPH and beta-End. High levels of beta-LPH and beta-End were also present in the plasma from a patient with the ectopic ACTH syndrome due to pancreatic carcinoma. Plasma immunoreactive 16-K fragment was increased in another patient with this syndrome. These results indicate that a biosynthetic model similar to that described in the AtT-20/D-16-v mouse tumor cell line also exists in man. Tumors responsible for the ectopic ACTH syndrome provide a unique source to study this model in man.

Human placental immunoreactive corticotropin, lipotropin, and beta-endorphin: evidence for a common precursor.

The concentrations and molecular sizes of immunoreactive corticotropin (ACTH), lipotropin (LPH, beta LPH plus gamma LPH), gamma LPH, and beta-endorphin (beta END) were determined in human placental extracts. Serial dilutions of a water extract of placenta generated competitive binding curves parallel with that of the standard in each assay. The concentrations of ACTH, LPH, gamma LPH, and beta END were 3.3, 0.8, 0.7, and 1.1 ng/g wet weight of tissue, respectively. A partially purified extract applied to a Sephadex G-50 column contained high Mr components with ACTH, LPH, gamma LPH, and beta END immunoreactivities. The extract was applied to an immune affinity chromatography column consisting of affinity-purified (1-24)ACTH antiserum covalently bound to agarose. The material that adsorbed to the column and eluted with buffer containing sodium dodecyl sulfate had ACTH, LPH, and beta END immunoreactivities, indicating that there was a component or components containing antigenic determinants for all of these peptides. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the affinity-purified placental extract revealed at least two high Mr components (Mr approximately 48,000 and 36,000) with all three immunoreactivities. These data suggest, but do not prove, that the placenta synthesizes ACTH, the LPHs, and beta END from a common precursor molecule.

Ectopic production of high molecular weight calcitonin and corticotropin by human small cell carcinoma cells in tissue culture: evidence for separate precursors.

The DMS-79 continuous line of human small cell lung carcinoma cells, which produces immunoreactive (IR)-corticotropin (ACTH), -lipotropin (LPH), and -beta-endorphin (beta END), was found to produce IR-calcitonin (CT). Two major high molecular weight (HMW) forms of IR-CT were observed after gel exclusion chromatography under denaturing conditions (mol wt. approximately 7,000 and approximately 14,000), as well as a minor HMW IR-CT component (mol. wt. approximately 70,000). None of these IR-CT materials was extracted from DMS-79 medium by affinity chromatography using an ACTH antibody covalently bound to agarose. These results demonstrate ectopic production of HMW forms of CT and ACTH/LPH/beta END by human lung tumor cells in tissue culture, but do not support the existence of a common CT/ACTH/LPH/beta END precursor molecule.

Corticotropin, lipotropin, and beta-endorphin production by a human nonpituitary tumor in culture: evidence for a common precursor.

A continuous line (DMS-79) of human pulmonary small cell carcinoma cells was shown to secrete immunoreactive adrenocorticotropin (ACTH), lipotropin, and beta-endorphin concomitantly into the culture medium. Gel filtration of the culture medium demonstrated at least five components: high molecular weight material(s) that had ACTH, lipotropin, and beta-endorphin immunoreactivities and materials similar to ACTH, beta-lipotropin, gamma-lipotropin, and beta-endorphin in their immunoreactivities and apparent molecular weights. The same components were observed when gel filtration was carried out in 6 M guanidine-HCl, and the high molecular weight material(s) appeared to consist of more than one component, with molecular weights in the range of 15,000-40,000. Immune affinity chromatography of the high molecular weight component(s) from gel filtration with a specific anti-(1-24)ACTH serum demonstrated that the ACTH, lipotropin, and beta-endorphin immunoreactivities were possessed by the same molecule(s), suggesting that ACTH, lipotropins, and beta-endorphin were derived from a common, high molecular weight precursor.

Method for preparation of ampoules containing microgram quantities of polypeptide hormones for distribution and use as laboratory standards: synthetic human ACTH and beta-MSH.

There are a number of polypeptide hormones, their subunits, and other derivatives that are available in such short supply as to preclude distribution by conventional methods to the many qualified investigators who require them. This report describes a method for preparing sealed ampoules of two such hormones, the utilization of which can provide many qualified investigators with comparable, stable reference compounds for several years and eliminate unnecessary waste and error associated with the distribution and use of bulk quantitites of peptide hormone preparations. Sealed ampoules of synthetic human adrenocorticotrophic hormone (alpha(h)(1-39) ACTH) and beta melanocyte-stimulating hormone (beta(h)(1-22) MSH) were prepared. Each ampuole contains 50 mug of dried and recoverable peptide plus 1 mg of lactose, sealed under dry nitrogen at atmospheric pressure. The respective preparations are fully active, both biologically and radioimmunologically, and are suitable as reference compounds in all types of biologic and radioimmunologic assays of these hormones. In addition, radioactive iodine labeling of each peptide can be accomplished without prior removal of the lactose.