Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.     

Purification and amino acid sequence of melanocyte-stimulating hormone from the dogfish Squalus acanthias

A melanocyte-stimulating hormone (MSH) was isolated by gel filtration and ion-exchange chromatography from extracts of the pituitary glands of dogfish. Sequence studies were carried out on the hormone and its enzymically and chemically cleaved fragments. The sequence of the hormone, Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Met, shows that ten of its 11 residues are the same as ten of the 13 residues of mammalian alpha-MSH. About half of its molecules have the carboxyl group at the C-terminus free and about half are amidated; about a fifth have an extra tyrosine residue on the N-terminus, thereby making 11 residues the same as in mammalian alpha-MSH. Unlike the mammalian hormone, however, none of it was found to be N-acetylated.     

Purification and amino acid composition of monomeric and polymeric collagens

The preparations and amino acid compositions of highly purified tropocollagen and insoluble polymerized collagen are described. These collagens appear to be very suitable for comparative studies in an investigation of the cross-linkages that are introduced into tropocollagen during the formation of polymerized collagen.     

Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus

Crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) are produced during the proteolytic processing of CHH preprohormones. Currently, the physiological roles played by CPRPs are unknown. Due to their large size, direct mass spectrometric sequencing of intact CPRPs is difficult. Here, we describe a novel strategy for sequencing Cancer productus CPRPs directly from a tissue extract using nanoflow liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Four novel CPRPs were characterized with the aid of MS/MS de novo sequencing of 27 truncated CPRP peptides. Extensive modifications (methionine oxidation and carboxy-terminal methylation) were identified in both the full-length and truncated peptides. To investigate the origin of the modifications and truncations, a full-length CPRP was synthesized and subjected to the same storage and extraction protocols used for the characterization of the native peptides. Here, some methionine oxidation was seen, however, no methylation or truncation was evident suggesting much of the chemical complexity seen in the native CPRPs is unlikely due to a sample preparation artifact. Collectively, our study represents the most complete characterization of CPRPs to date and provides a foundation for future investigation of CPRP function in C. productus.     

Occurrence of L- and D-crustacean hyperglycemic hormone isoforms in the eyestalk X-organ/sinus gland complex during the ontogeny of the crayfish Astacus leptodactylus.

We studied the ontogeny of the eyestalk structure and of the L-CHH and d-Phe3-CHH synthesis in the X-organ/sinus gland (XO/SG) complex by light microscopy and immunocytochemistry in the freshwater crustacean Astacus leptodactylus. The optic ganglia start to differentiate in embryos at EI 190 microm (EI: eye index; close to 410 microm at hatching). At EI 270 microm, the three medullae (externa, interna, and terminalis) and the lamina ganglionaris are present and are organized as in the adult eyestalk. The L-CHH was localized in perikarya of neuroendocrine cells, in their tracts, and in SG from the metanauplius stage to the adult. The d-Phe3-CHH was visualized in XO perikarya, in their tracts and in SG of embryos from EI 350 microm and in all later studied stages. Co-localization of both CHH stereoisomers always occurred in the d-Phe3-CHH-producing cells. These results show that the synthesis of CHH enantiomers starts during the embryonic life in A. leptodactylus, and that the d-isomer is synthesized later than its L-counterpart. We discuss the post-translational isomerization as a way to generate hormonal diversity and the putative relation between d-Phe3-CHH synthesis and the ability to osmoregulate, occurring late during the embryonic life of Astacus leptodactylus.     

Adrenocorticotropin 53. The amino acid sequence of the hormone from the ostrich pituitary gland.

The amino acid sequence of corticotropin from the ostrich pituitary gland has been determined. It consists of 39 amino acids with the following sequence: H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Arg-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Val-Gln-Glu-Glu-Thr-Ser-Glu-Gly-Phe-Pro-Leu- Glu-Phe-OH. This is the first report on the primary structure of corticotropins from avian species.     

Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.     

The role of the Y-organ and cephalic gland in ecdysteroid production and the control of molting in the crayfish,Orconectes limosus

Summary The production of ecdysteroids (monitored by RIA) by Y-organs and cephalic glands in vitro was measured and hemolymph ecdysteroid levels were determined in the crayfish,Orconectes limosus, both after eyestalk ablation and as a function of time during natural premolt. Y-organ synthesis of ecdysteroid increased in parallel with a rise in hemolymph ecdysteroid concentrations under both conditions, peaking in substage D₂ of premolt. Y-organ ecdysteroid output after eyestalk ablation was 3–4 times higher. Thus, removal of the inhibiting system of the eyestalk effectively removes not only the principal control but also any modulation of ecdysteroid secretion by the Y-organs. Ecdysteroid levels remained low in Y-organ-ectomized crayfish, although premolt was initiated in some animals. The cephalic gland does not appear to contribute to the regulation of molting inOrconectes limosus. The Y-organs, on the other hand, are a principal source of ecdysteroids which regulate the major synthetic activities of premolt.     

Variation in amino acid composition of legumin from Pisum

Pisum; Leguminosae: pea: amino acid composition: seed storage proteins: legumin.     

Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas,eriocheir sinensis,andUca pugilator

Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.     

Purification,characterisation and amino acid composition of the putative moult-inhibiting hormone (MIH) ofCarcinus maenas (Crustacea,Decapoda)

Summary Using a Y-organ in vitro assay to measure repression of ecdysteroid synthesis in the presence of putative moult-inhibiting hormone (MIH), in conjunction with HPLC separation of sinus gland neuropeptides ofCarcinus maenas, it was found that both the hyperglycemic hormone (CHH) and a novel peptide (argued to represent the MIH) inhibited ecdysteroid synthesis. The latter was purified to homogeneity, and amino acid analysis showed that it is a 61 residue peptide (minimum molecular mass 7,200 Da) with the following amino acid composition: Asx₉; Thr₂; Ser₂; Glx₇; Pro₁; Gly₄; Ala₂; 1/2 Cys₄; Val₄; Met₁; Ile₃; Leu₅; Tyr₁; Phe₃; His₃; Trp₂; Lys₂; Arg₆. The N-terminus appears to be blocked. MIH is at least 20 times more potent than CHH in repressing ecdysteroid synthesis and is active at concentrations of less than 250 pmol/l. There may be structural similarities between CHH and MIH, howeve, MIH displays no CHH radioimmunoreactivity or hyperglycemic activity. The physiological significance of CHH in controlling ecdysteroid titres is not known.Abbreviations CHH hyperglycemic hormone - MIH moult inhibiting hormone - PAGE polyacrylamide gel electrophoresis - RIA radioimmunoassay - SDS sodium dodecyl sulfate - SG smus gland(s) - SGE sinus gland equivalent - TFA trifluoroacetic acid     

Purification and characterization of LPXTGase from Staphylococcus aureus: the amino acid composition mirrors that found in the peptidoglycan

Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from Streptococcus pyogenes that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from Staphylococcus aureus and found that it has both similar and unique features compared to the S. pyogenes enzyme. The S. aureus enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alanine, and lysine, and furthermore, we find that these amino acids are present in the enzyme at precisely the same ratio at which they are found in the peptidoglycan for the respective organism. This suggests that enzymes responsible for wall assembly may also play a role in the construction of LPXTGase.