A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.

Based on modifications to enhance the a-helical structure of the broad spectrum antibiotic magainin 2, a series of analogues have been synthesized which display an increase up to two orders of magnitude in antimicrobial activity and, in the most favorable case, no appreciable increase in hemolytic activity over magainin 1 at the concentrations tested.

We have previously reported the isolation of two broad-spectrum antimicrobial peptides ("magainins") from the skin of the African clawed frog Xenopus laevis. These natural peptides are active against many species ofbacteria and fungi and also induce osmotic lysis of protozoa. In this report we demonstrate that synthetic magainin peptides appear to be indistinguishable from the natural products with respect to chromatographic properties and biological activity. These studies demonstrate conclusively that the magainin peptides are potent antimicrobial substances.We have previously reported the isolation of two broadspectrum antibiotic peptides called "magainins" from the skin of the African clawed frog Xenopus laevis (1 In this report we demonstrate that the exceptionally broad range of antimicrobial activity exhibited by the magainin peptides recovered from Xenopus skin can be reproduced with synthetic analogues of the corresponding peptides. We show that the natural and synthetic peptides appear to be indistinguishable with respect to both biological and chromatographic properties. In addition, through the synthesis of several truncated analogues, we provide some insight into the structural requirements for the antimicrobial activity of the magainin peptides.MATERIALS AND METHODS Purification of Magainin Peptides. Magainin peptides were purified from the skin of the ventral surface of a female X. laevis as previously described (1).Assay of Magainin Antibacterial Activity. The standard assay has been described (1).Amino Acid Sequencing. Peptides were sequenced on an Applied Biosystems gas-phase protein sequencer (model 470A; Applied Biosystems, Foster City, CA) by the Edman degradation procedure. A standard version 3.0 program was followed, and all reagents used in the instrument were supplied by the manufacturer.Carboxyl-Terminal Analysis of Peptides. Between 2 and 4 nmol of peptide was incubated with 1 ,g of carboxypeptidase Y (122 units/mg; Sigma) in 55 ,ul of 0.1 M pyridine/acetate (pH 5.5) for 15 min at 21°C. Reactions were stopped by the addition of 50 .ul of 1 M acetic acid, and the solutions were then lyophilized to dryness. Amino acids were subsequently analyzed on a Beckman model 121 MB analyzer.Solid-Phase Peptide Synthesis. Peptides were synthesized on an Applied Biosystems peptide synthesizer using the manufacturer's reagents. Deprotection and cleavage from the resin by anhydrous HF followed standard procedures (3, 4). Methionine sulfoxides were reduced by incubation of the peptides in the presence of dithiothreitol (5). Peptides were purified to homogeneity by HPLC as described in Fig. 1. RESULTS As we have reported previously, several chromatographically distinct peptides with antimicrobial activity can be recovered from Xenopus laevis skin (1). The purification that resulted in successful recovery of antimicrobial activity involved homogenization of the skin of an anesthetized female Xenopus in acetate buffer at pH 4.0, step elution chromatography on carboxymethylcellulose, followed by s...

The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins [Rnh2Ap (Rnh201p), Ydr279p (Rnh202p) and Ylr154p (Rnh203p)] that together are necessary and sufficient for RNase H2 activity [correction]. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordicn charuntia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (ii) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-l infected H9 cells. The doses required for 50% inhibition (ID& in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-l infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined.

No abstract

No abstract

No abstract