Johannes Meienhofer

Preparative high-performance liquid chromatography applied to peptide synthesis☆

A variety of protected synthetic peptides were purified by high-performance liquid chromatography on pre-packed silica gel columns. The compounds varied in protecting groups, amino acid composition and molecular weight. Flow-rates of two to four column volumes per hour were employed, with resultant back pressure of less than 150 p.s.i. A typical column load of 0.2-5 g was purified in 2-5 h.

Peptide E: Opiate receptor binding profile in the rat brain membrane assay. Comparison with beta-endorphin

Beta-endorphin (beta-EP) and peptide E were compared in respect to their binding potency in the rat brain membrane by radioreceptor binding assay using tritiated human beta-EP, [D-Ala2,D-Leu5]-enkephalin (DADLE), dihydromorphine (DHM) and ethylketocyclazocine (EKC) as primary ligands. When the potency of beta h-EP was chosen to be 100%, peptide E was equipotent with beta-EP in displacing DHM (95%) and EKC (103%) less potent for competing with beta h-EP (60%) and least active (7%) for displacing DADLE. It may be concluded that peptide E binds preferentially with the opiate mu and kappa receptors in the rat brain membrane.

Synthesis of adrenal peptide E and some of its biological activities

A synthesis of peptide E, a highly potent, 25-amino acid adrenal opioid peptide containing both a [Met]enkephalin at the NH2-terminus and [Leu]enkephalin sequence at the COOH-terminus, originally isolated from bovine adrenal medulla [D. L. Kilpatrick, T. Taniguchi, B. N. Jones, A. S. Stern, J. E. Shively, J. Hullihan, S. Kimura, S. Stein, and S. Udenfriend (1981) Proc. Natl. Acad. Sci. USA 78, 3265-3268], is reported. The synthesis was accomplished by the solid-phase method employing the 4-(aminoacyloxymethyl)phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. Two synthetic strategies (N-indole formyl protected vs unprotected tryptophan) were followed and results compared and evaluated. It was determined that peptide E prepared with protection of tryptophan (residues 13 and 14) was preferred and gave final product that was readily purified by HPLC. The biological activity of the synthetic material was found to be equivalent to the reported activity of the natural compound.

The gastrointestinal absorption, tissue distribution, urinary excretion and metabolism of N-(2-aminoethyl)-glycine (AEG) in the rat

Abstract Radiolabeled N-(2-aminoethyl)-glycine (AEG) was synthesized and various aspects of its bioavailability were evaluated. AEG was rapidly and completely taken up by the small intestine of the rat. It was quickly absorbed into the portal vein. Most of the absorption took place during the first hour, with a peak at 30 min. Entry of this compound into the intestinal mucosal cell may be by a mechanism not involving active transport. Of many organs examined, only the liver took up significant amounts of AEG. The latter neither crossed the brain barrier nor was metabolized. Total urinary excretion (as intact AEG) averaged 80% of the administered dose within 4 hours and nearly 100% by 10 hours. Excluding the neutral-acidic amino acids and ammonia, AEG represented >99% of the ninhydrin positivity in the urine. AEG is thus an example of a substance which is rapidly and totally absorbed, as well as quickly and completely excreted.

Isolation of Thymosin α1 from Thymosin Fraction 5 by High Performance Liquid Chromatography

Abstract An HPLC system for quickly isolating individual peptides from thymus extract is described. A series of reversed phase separations followed by ion-exchange HPLC produces pure peptides with minimal chromatographic time. A 2.4 gram column load of crude extract was carried through to the isolation of 4 mg of pure thymosin α1 within 6–8 hours of chromatography.

Chapter 17: Peptide Hormones

Publisher Summary Several years of search for a putative factor in brain, which activates opiate receptors, has culminated in the isolation of two pentapeptides, Met-enkephalin [Ia] and Leu-enkephalin [Ib] from guinea pig brain. Both have been synthesized and showed typical opioid activity in vitro and analgesic effects in vivo . This chapter reviews Peptide hormones that are of other than hypothalamic or pituitary origin. The structures of seven species-different calcitonins have been determined. Conventional syntheses of porcine, human and salmon calcitonin provided large supplies of homogeneous peptides for physiological, pharmacological, and clinical investigations. Salmon calcitonin is far more potent in man than human calcitonin that may be attributable to increased metabolic stability. Secretion of calcitonin from porcine thyroid glands is effected by activation of C-cell adenyl cyclase by either ionized Ca or by hormones (glucagon, gastrin, CCK, epinephrine). Calcitonin effects are age-dependent and are very low in normal adult humans. 6-Lipotropin is thus another example of a second order prohormone, that is, a well-defined peptide hormone serving as a precursor for a smaller peptide with entirely different hormonal activities. The characterization of the opioid peptides may suggest new approaches to the development of nonaddictive analgesics.

Chapter 21. Peptide Hormones of the Hypothalamus and Pituitary

Publisher Summary This chapter describes the biological activities and therapeutic application of peptide hormones of the hypothalamus, pituitary, and neurohypophyseal hormones. The hypothalamus hormones discussed are gonadotropin releasing hormone (GNrH), melanotropin release-inhibiting factor (MIF) and melanotropin releasing factor (MRF), somatostatin, and thyrotropin releasing hormone (TRH). GnRH might be expected to find diagnostic, prophylactic, and therapeutic applications in human and animal fertility. One problem facing in vivo applications is the short duration of action of GnRH. The significance and physiological roles of peptides, which exhibit H-Pro-Leu-Gly-NH2 (MIF) or MRF activities are still subjects of debate because of great experimental difficulties with bioassays. Concerning biosynthetic aspects, however, the enzymatic formation of MIF from oxytocin was the first example, on a molecular basis, of a well-defined peptide hormone serving as a precursor or second order prohormone for a smaller peptide with entirely different hormonal activities. The pituitary hormones discussed in this chapter are adrenocorticotropin and melanotropin, growth hormone and prolactin, and glycoprotein hormones (follicle stimulating hormone, luteinizing hormone, and thyroid stimulating hormone).

The Synthesis and Physical Properties of Thymosin α1 and Its Analogs Prepared by Fragment Condensation

The isolation of biologically important peptides from the thymus gland has been studied extensively in the last few years. Several thymic peptides have been shown to play certain roles in T-cell maturation (White, 1980; Trainin et al., 1980a, b; Goldstein and Lau, 1980; Bach and Goldstein, 1980). Thymosin α1, a highly acidic Nα-acetyl octacosapeptide, isolated from calf thymus gland (Goldstein et al., 1977) and characterized by sequence analysis (Low and Goldstein, 1979), has been reported to exhibit biological activities involved in the development of thymus-dependent lymphocytes (T cells) (Goldstein et al., 1977). Thymosin a1 has been synthesized by classical procedures in solution (Wang et al., 1979; Birr and Stollenwerk, 1979) and by solid-phase methods (Wong and Merrifield, 1980; Wang et al., 1980; Colombo, 1981).

Fluorometric-colorimetric amino acid analysis of actinomycins using fluorescamine: Mechanism of partial threonine decomposition during acid hydrolysis

Abstract a fluorometric-colorimetric fluorescamine analyzer was successfully utilized for the analysis of a series of actinomycin hydrolysates containing N -methyl-amino acids, proline, and proline derivatives. An explanation is proposed for the low threonine values consistently observed in the analysis of actinomycin hydrolysates and alternate conditions for improved hydrolysis of actinomycins are proposed.