Anthony J. Mason

Structure of two human ovarian inhibins

The complete amino acid sequences of two forms of human ovarian inhibin have been determined through cloning and nucleotide sequencing of cDNAs encoding their individual subunit precursors. The alpha subunit common to both forms of human inhibin is homologous (84 percent conserved) to its equivalent porcine alpha subunit; the subunits which are different in both inhibins (beta A and beta B) are identical to their porcine equivalents in all but one of 232 sequence positions.

A prolactin-inhibiting factor within the precursor for human gonadotropin-releasing hormone

The cloned complementary DNA sequence encoding the human gonadotropin-releasing hormone (GnRH) precursor protein was used to construct an expression vector for the bacterial synthesis of the 56-amino acid GnRH-associated peptide (GAP). GAP was found to be a potent inhibitor of prolactin secretion and to stimulate the release of gonadotropins in rat pituitary cell cultures. Active immunization with peptides corresponding to GAP sequences led to greatly increased prolactin secretion in rabbits.

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a λgt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N‐terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy‐terminus, located extracellularly.

Molecular cloning and amino acid sequence of rat enkephalinase

cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.

The Mammalian GnRH Gene and Its Pivotal Role in Reproduction

Publisher Summary The key regulatory brain peptide controlling reproduction in mammals and sub-mammalian vertebrate species is the decapeptide luteinizing hormone-releasing hormone (LHRH), also called gonadotropin-releasing hormone (GnRH). This peptide, synthesized in and secreted from hypothalamic neurosecretory cells, stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitary gonadotrophs. The miniscule amounts of GnRH in hypothalamus as well as its modified N- and C-termini made its isolation and structural determination a monumental task. This chapter explains the structure and restriction map of the human and rat GnRH-GAP gene. The description of the gene structure and the GnRH precursor sequence has aided in the investigation of GnRH gene expression in various parts of the mammalian organism. Using mainly immunological procedures, it has been found that GAP and GnRH are produced in a surprisingly large number of tissues. They occur in varying amounts in placenta, mammary tissue, gonads, kidney, as well as in certain extra-hypothalamic parts of the central nervous system.

Purification and characterization of recombinant human activin B

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the beta subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the beta subunit of inhibin B. Activin B has an apparent pI of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone

The secretion of follicle-stimulating hormone (FSH) by pituitary cells is stimulated by activin and gonadotropin-releasing hormone, GnRH. To examine the possible interrelationships between the intracellular actions of these secretagogues, responsiveness to activin was tested following pretreatment with 0, 0.1, or 10 nM GnRH. In cells pretreated with 0 or 0.1 nM GnRH, FSH secretion was increased approximately 2-fold during a subsequent challenge with either activin or GnRH. In contrast, in cells pretreated with 10 nM GnRH, FSH secretion became unresponsive to GnRH but could still be stimulated 2-fold by activin. These results demonstrate that activin is able to stimulate FSH secretion in cells that have undergone desensitization to GnRH.

Gonadal development and gametogenesis in the hypogonadal mouse are restored by gene transfer.

These results describe controlled regulation of a mammalian neural gene in transgenic mice. Analysis of truncated GnRH-GAP genes in transgenic mice will enable us to define the DNA sequences responsible for this control. Furthermore, by separate mutation of the GnRH and GAP coding sequences we will be able to determine the relative importance of these two peptides in the development and maintenance of reproductive function.

The gene encoding GnRH and its associated peptide GAP: Some insights into hypogonadism☆

The hypogonadal (hpg) mouse represents a unique animal model for hypogonadism. In this mutant the truncation of the gene encoding GnRH and its associated peptide GAP leads to drastically lowered gonadotropin levels and increased circulating prolactin. This deficiency in turn leads to a failure of testes and ovaries to develop normally. Using gene therapy we have restored the reproductive functions of the hpg mouse. The success of this therapy uniquely underscores the importance of the gene encoding the GnRH precursor and lends credence to the hypothesis that no other gene in mammals can replace it. As a consequence, defects in the control and/or structural properties of the human GnRH are expected to result in hypogonadism in humans.

Localization of Activin βA-,βB-, andβC-Subunits in Human Prostate and Evidence for Formation of New Activin Heterodimers ofβC-Subunit1

Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.