Partial purification and characterization of a peptide with growth hormone-releasing activity from extrapituitary tumors in patients with acromegaly.

Frohman, Lawrence A.; Szabó, Márta; Berelowitz, Michael; Stachura, Max E.

doi:10.1172/jci109658

Cited by 165 publications

(47 citation statements)

References 35 publications

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“…Single injection protocol. After obtaining a basal sample, subjects were given a rapid intravenous injection of hpGRF Bioassay of GRF GRF activity of plasma extracts was measured in a dispersed rat pituitary cell monolayer culture system as previously described (17,18), which was modified by using serum-free media supplemented with bovine insulin, 10 ,g/ml, and corticosterone, 10-8 M (Sigma Chemical Co., St. Louis, MO), during the 4 d before testing. Growth hormone release was measured during a 4-h incubation period in quadruplicate cultures.…”

Section: Methodsmentioning

confidence: 99%

Metabolic clearance and plasma disappearance rates of human pancreatic tumor growth hormone releasing factor in man.

Frohman¹,

Thominet²,

Webb³

et al. 1984

J. Clin. Invest.

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Abstract. The metabolic clearance rate (MCR) and plasma disappearance rate (t1/2) ofhuman pancreatic tumor growth hormone releasing factor ] was determined in normal adult male subjects by single injection and constant infusion techniques. Single injections of 1, 3.3, and 10 ,Lg/kg hpGRF(l-40) were administered intravenously, plasma immunoreactive (IR) GRF levels were measured during the subsequent 180 min, and biexponential curve analysis was performed. Graded, dose-constant infusions of hpGRF(1-40) at rates of 1, 3.3, 10, and 33 ng/kg per min were administered and the MCR was calculated from measurement of steady state plasma IR-GRF levels at each infusion rate. The postinfusion disappearance rate was determined by linear regression analysis of plasma IR-GRF levels during the 120-min period after cessation of the infusion.The calculated MCR during the single injection study was 194±17.5 liters/M2 per d and was not significantly different from the calculated value during the constant infusion study (202±16 liters/M2 per d). The disappearance rate during the single injection study was subdivided into two linear phases: an initial equilibration phase (7.6±1.2 min) and a subsequent elimination phase (51.8±5.4 min). The latter was similar to the linear disappearance rate observed (41.3±3.0 min) after cessation of the constant infusion. The chromatographic and bi-

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Section: Methodsmentioning

confidence: 99%

Metabolic clearance and plasma disappearance rates of human pancreatic tumor growth hormone releasing factor in man.

Frohman¹,

Thominet²,

Webb³

et al. 1984

J. Clin. Invest.

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“…Growth hormone (GH)l-releasing hormone (GRH) was first identified and sequenced in tumors from which it was ectopically secreted (1)(2)(3). Two forms ofGRH, GRH( 1-40)-OH and GRH(l-44)-NH2, that have been identified from both tumors and hypothalamus, are identical in structure (4), exhibit indistinguishable biologic activity, and are derived from a single precursor (5).…”

Section: Introductionmentioning

confidence: 99%

Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus.

Frohman¹,

Downs²,

Williams³

et al. 1986

J. Clin. Invest.

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147

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The effect of plasma on degradation of human growth hormonereleasing hormone (GRH) was examined in vitro and in vivo using high performance liquid chromatography (HPLC), radioimmunoassay (RIA), and bioassay. When GRH(1-44)-NH2 was incubated with human plasma, the t1/2 of total GRH immunoreactivity was 63 min (RIA). However, HPLC revealed a more rapid disappearance (t1/2, 17 min) of GRH(144)-NH2 that was associated with the appearance of a less hydrophobic but relatively stable peptide that was fully immunoreactive. Sequence analysis indicated its structure to be GRH(344)-NH2. Identity was also confirmed by co-elution of purified and synthetic peptides on HPLC. Biologic activity of GRH(344)-NH2 was <10-3 that of GRH(144)-NH2. After intravenous injection of GRH(1-44)-NH2 in normal subjects, a plasma immunoreactive peak with HPLC retention comparable to GRH(344)-NH2 was detected within 1 min and the tl/2 of GRH(144)-NH2 (HPLC) was 6.8 min. The results provide evidence for GRH inactivation by a plasma dipeptidylaminopeptidase that could limit its effect on the pituitary.

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“…Many investigators have reported the presence of GH-releasing activity in human carcinoid tumors (13-15) and pancreatic islet cell tumors removed from patients with acromegaly (15)(16)(17). Recently, human pancreatic GRF (hpGRF) was isolated and structurally characterized from two pancreatic islet cell tumors in patients with acromegaly (9,18,19).…”

mentioning

confidence: 99%

“…In addition, the NH2-terminal 29 amino acids of hpGRF maintain full intrinsic activity and potency in vitro (18), and recently amino acids [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] tion was stirred on a rotary mixer for 1 hr at room temperature, then dialyzed in spectapor 3 membrane tubing (Spectrum Medical Industries, Los Angeles) against a 1-liter 10 mM phosphate buffer (pH 7.4) for 2 hr at room temperature with constant stirring. The dialyzed complexes were emulsified with complete Freund's adjuvant (GIBCO) and normal saline (1:1:1) in the presence of 10 mg of active charcoal (Fisher), using two glass syringes connected by a three-way stopcock until the emulsion was firm.…”

mentioning

confidence: 99%

Levels of human and rat hypothalamic growth hormone-releasing factor as determined by specific radioimmunoassay systems.

Audhya¹,

Manzione²,

Nakane³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Polyclonal antibodies to synthetic human pancreatic growth hormone-releasing factor )NH2] and rat hypothalamic growth hormone-releasing factor [rhGRF(1-43)OHJ were produced in rabbits by injecting these weak immunogens, coupled to thyroglobulin and emulsified with complete Freund's adjuvant in the presence of activated charcoal, directly into the spleen. A subsequent booster in-jection by the conventional intramuscular route resulted in high-titer antibodies, which at a 1:20,000 dilution were used to develop highly sensitive and specific radioinmunoassays for these peptides. By using antibodies with an apparent K. of 3.3x 10-12 (human) and 7.7 x 10-11 (rat), the sensitivity of these assays in both human and rat was found to be <1 fmol. The antibody to hpGRF(1-44)NH2 is directed against the COOHterminal region of the molecule, as shown by its crossreactivity with various hpGRF analogues: 140% with hpGRF(30-44)NH2; 1%-2% with hpGRF(1-37)OH, hpGRF(140)OH, and hpGRF(1-40)NH2; and none with hpGRF(1-29)NH2. Serial dilutions of human and rat hypothalamic extracts demonstrated parallelism with the corresponding species-specific standard and 12,5-labeled tracer. There was no crossreactivity with other neuropeptides, gastrointestinal peptides, or hypothalamic extracts of other species. The hypothalamic content in fmol/mg (wet weight) of tissue was 3.6 ± 0.2 for the human and 11.1 ± 5.5 for the rat. Age-related changes in hypothalamic GRF content were present in rats, with a gradual increase from 2 to 16 weeks and a correlation between increasing body weight and GRF content. These radioimmunoassays winl serve as important tools for understanding the regulation of growth hormone secretion in both human and rat.Since 1959 (1), it has been known that the hypothalamus is involved in the control of growth hormone (GH) secretion. Experimental and clinical evidence has since indicated that the hypothalamus performs a dual function in the regulation of GH secretion from the anterior pituitary (2-4). Inhibition of GH release is accomplished by an inhibitory peptide, somatostatin (SRIF), which in 1972 was isolated from ovine hypothalami, purified, and characterized as a tetradecapeptide (5). In contrast, as suggested by physiological evidence (2-4) and GH-releasing activity in hypothalamic extracts (6-8), stimulation of GH release is accomplished by a stimulatory peptide, GH-releasing factor (GRF). Until 1982, howev-er, the isolation and characterization of GRF remained elusive. The difficulty in its characterization had been due to the minute quantities of the peptide in hypothalamic tissue, the large quantities of SRIF that interfere in the bioassay, and the fact that the methodology used for peptide sequencing and purification has only recently become available (9-12).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2970Many investigators have reported the presenc...

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Partial purification and characterization of a peptide with growth hormone-releasing activity from extrapituitary tumors in patients with acromegaly.

Cited by 165 publications

References 35 publications

Metabolic clearance and plasma disappearance rates of human pancreatic tumor growth hormone releasing factor in man.

Metabolic clearance and plasma disappearance rates of human pancreatic tumor growth hormone releasing factor in man.

Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus.

Levels of human and rat hypothalamic growth hormone-releasing factor as determined by specific radioimmunoassay systems.

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