Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency.

The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species.

Isolation and amino acid sequence of a peptide with vitellogenesis inhibiting activity from the terrestrial isopod Armadillidium vulgare (Crustacea).

The complete amino acid sequence of a neuropeptide was established using gas-phase microsequencing, mass spectrometry, and reverse transcription-polymerase chain reaction. This peptide, stored in the sinus gland of the terrestrial isopod Armadillidium vulgare, inhibited vitellogenin synthesis by the fat tissue and inhibited the onset of secondary vitellogenesis when tested in homologous bioassays. This peptide, named Arv-VIH, has 83 amino acid residues and a molecular mass of 9485 Da.

Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough.

Lactobacillus plantarum ST31 isolated from sourdough produced an antimicrobial substance inhibiting other strains of the genera Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Bacillus and some foodborne pathogens including Staphylococcus aureus. This antimicrobial substance was inactivated by proteolytic enzymes. Consequently, it was characterized as a bacteriocin and was designated plantaricin ST31.

The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare.

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC-purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino-acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N-glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges.

Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem.

Aah VI, a novel, N-glycosylated anti-insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination.

Aah VI was isolated from the venom of the North African scorpion, Androctonus australis hector. It is the first glycosylated neurotoxin from scorpion venom to be described. It was not toxic to mice, when injected intracerebroventricularly at a dose of 1.

The chemically inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics.

Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas.

Divercin V41, a new bacteriocin with two disulphide bonds produced by Carnobacterium divergens V41: primary structure and genomic organization.

Divercin V41 is a new bacteriocin produced by Carnobacterium divergens V41, a lactic acid bacterium isolated from fish viscera. The amino acid sequence of divercin V41 showed high homologies with pediocin PA-1 and enterocin A. Two disulphide bonds were present in the hydrophilic N-terminal domain and in the highly variable hydrophobic C-terminal domain, respectively.

Phosphorylation and O-glycosylation sites of bovine chromogranin A from adrenal medullary chromaffin granules and their relationship with biological activities.

Bovine adrenal medullary chromogranin A, the major soluble component of chromaffin granules, is a phosphorylated glycoprotein. In the present work, phosphorylation and glycosylation sites were determined using mild proteolysis, peptide separation, microsequencing, and mass analysis by electrospray and matrix-assisted laser desorption ionization time-of-flight techniques. Seven post-translational modification sites were detected.

Specific proteolytic cleavage of the myristoylated alanine-rich C kinase substrate between Asn 147 and Glu 148 also occurs in brain.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major ubiquitous substrate of protein kinase C. The expression of the protein is regulated during cell cycle progression and cell proliferation. Specific proteolytic cleavage of the protein between Asn 147 and Glu 148 was described recently in cultured cells, and the corresponding proteolytic activity was observed in various tissue extracts except for brain.

Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific inhibitory activity.

Two bacteriocins produced by Carnobacterium piscicola V1 were purified and characterized. Piscicocin V1a (molecular mass = 4,416 Da) and piscicocin V1b (molecular mass = 4,526 Da) are nonlantibiotic, small, heat-stable antibacterial peptides. Piscicocin V1b is identical to carnobacteriocin BM1, while piscicocin V1a is a new bacteriocin.

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin.

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone.

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM-resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the anti-native neurohormone serum and the same biological activity as the native neurohormone.

Solution structure of PMP-D2, a 35-residue peptide isolated from the insect Locusta migratoria.

The three-dimensional solution structure of PMP-D2, a 35 amino acid peptide isolated from the insect Locusta migratoria, has been determined from two-dimensional 1H NMR spectroscopy data. The structure calculations were performed from 222 NOE-derived interproton distances and 11 dihedral angles calculated from the JHN-H alpha coupling constants, using either a combination of distance geometry and restrained simulated annealing or by restrained simulated annealing alone. PMP-D2 contains three disulfide bridges that have been assigned from NMR data and structure calculations and independently confirmed using chemical and enzymatic methods.

Demyristoylation of the major substrate of protein kinase C (MARCKS) by the cytoplasmic fraction of brain synaptosomes.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a major substrate of protein kinase C, whose interaction with the plasma membrane is dependent on its phosphorylation by protein kinase C and on its N-terminal myristoylation. We describe a hitherto undescribed demyristoylation activity of the protein in cytoplasmic fraction of synaptosomes from bovine brain. The activity is dependent on ATP but independent from calcium.

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study.

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state.

Sequence of pig lens aldose reductase and electrospray mass spectrometry of non-covalent and covalent complexes.

The complete sequence of pig lens aldose reductase (EC 1.1.1.

Synthesis and characterization of kaliotoxin. Is the 26-32 sequence essential for potassium channel recognition?

Kaliotoxin (KTX), a scorpion toxin characterized as a 37-residue inhibitor of the neuronal high conductance Ca(2+)-activated K+ channels (KCa channels), has been chemically synthetized. Differences were observed between natural toxin and the two peptides, KTX(1-37) and KTX(1-37)-amide. Re-examination of the KTX sequence showed that an extra lysine residue was present at the C-terminal end.

Isolation of the non-myristoylated form of a major substrate of protein kinase C (MARCKS) from bovine brain.

A substrate protein of protein kinase C with an apparent molecular mass of 70 kDa has been purified from bovine brain. This protein shares several properties with a major substrate of protein kinase C (myristoylated alanine-rich C kinase substrate; MARCKS). It is heat-stable and copurifies with MARCKS during various steps (ammonium sulfate precipitation and gel filtration).

Isolation and molecular characterization of a hyperglycemic neuropeptide from the sinus gland of the terrestrial isopod Armadillidium vulgare (Crustacea).

The major peptide from the sinus gland of the terrestrial isopod Armadillidium vulgare (Crustacea) has been extracted and purified by reverse-phase HPLC. This neuropeptide exhibited a high hyperglycemic activity and was therefore named A. vulgare crustacean hyperglycemic hormone (Arv-CHH).

Affinity purification and characterization of myristoylated alanine-rich protein kinase C substrate (MARCKS) from bovine brain. Comparison of the cytoplasmic and the membrane-bound forms.

A major in vivo substrate of Ca(2+)-phospholipid-dependent protein kinase (myristoylated alanine-rich C-kinase substrate (MARCKS)) has been purified to apparent homogeneity from the particulate as well as from the cytoplasmic fractions of calf brain using a calmodulin affinity column. The two preparations were characterized and compared with various biochemical and biophysical techniques. Although they behave similarly in various chromatographic procedures during purification, their elution positions from the gel filtration column are markedly different.

Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin.

Purification and characterization of androgenic hormone from the terrestrial isopod Armadillidium vulgare Latr. (Crustacea, Oniscidea).

Androgenic hormone (AH) was purified from hypertrophied androgenic glands of intersexed Armadillidium vulgare (genetic males feminized by symbiotic endocellular bacteria). Two isohormones labeled AH1 and AH2 with similar molecular weights in the range 17,000-18,000 were isolated. Amino acid analysis showed the absence of cysteine in these two forms.