Microsequence analysis of peptides and proteins: trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis

A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 N HCl (30 min at room temperature) was used for cleavage as originally described by G.R. Stark (Biochemistry 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90 degrees C were 5.6 X 10(-5), 5.1 X 10(-4), and 8.6 X 10(-4) s-1, respectively. In order to obtain complete couplings within 30-40 min at 50 degrees C, the effect of pyridine catalysis was studied. The addition of 0.225 M pyridine resulted in roughly doubling the rates at 50 and 70 degrees C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50 degrees C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.     

Is the initiation of macronuclear DNA synthesis in Euplotes dependent on micronuclear functions?

To determine whether the micronucleus makes essential contributions during asexual reproduction, observations were made on cells of Euplotes octocarinatus from which the micronucleus had been removed with a micropipette. Most cells underwent one postenucleation division, then became arrested in macronuclear G1, slowed down in food uptake, developed macronuclear deformations, and finally died. Such cells could be rescued if a micronucleus was reimplanted before macronuclear deformations had developed. When provided with a new micronucleus, cells initiated macronuclear DNA synthesis about 12-16 h later. The data suggest that the micronucleus is involved in the control of the cell's transition from macronuclear G1 to S, and a model is proposed which postulates that in Euplotes macronuclear DNA synthesis is initiated when a micronucleus-encoded "initiator protein" has accumulated to a critical amount.     

Beobachtungen zur Lebensweise von Pycnogonum litorale (Ström) (Pantopoda)

Zusammenfassung Am Leitdamm des Jadebusens lebt Pycnogonum litorale im Lückensystem des Miesmuschelbesatzes. Dieser bietet mit hartem Untergrund, hoher Feuchtigkeit bei Niedrigwasser, genügend Actinien als Nahrung und guter Durchströmung bei gleichzeitigem Schutz vor Vertragung—offensichtlich günstige Lebensbedingungen für Pycnogonum litorale.Der Eiablage im Februar geht eine Reiterstellung des Männchens auf dem Weibchen von durchschnittlich 24 Tagen voraus. Unter künstlichen Kurztagbedingungen kann diese Reiterstellung auch außerhalb der Fortpflanzungsperiode eingenommen werden. Die Eier werden durch Rumpfbewegungen beider Partner zu den Ovigeren des Männchens bewegt. Bei 12°C schlüpfen die Larven etwa 41 bis 46 Tage nach der Eiablage aus, bei 19°C, im Sommer, schlüpften keine Larven.Im Jadebusen leben die Larven etwa 1/2 Jahn endoparasitisch in Hydrozoen. Die an die Metamorphose anschließende juvenile Phase, in der die Tiere frei leben, dauert ein knappes Jahr, die Reifehäutung erfolgt normalerweise im Sommer des zweiten Jahres, die Fortpflanzungsperiode etwa 6 Monate später, im Winter.

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Observations on the life biology of Pycnogonum litorale (Ström) (Pantopoda)

Summary Pycnogonum litorale lives in an interstitial system, of the mussel zone on the embankment of the Jadebusen. Hard substrate, high humidity at low tide, sufficient Metridium senile as food, and active currents together with protection from drifting, constitute favourable conditions for this pycnogonid.Prior to laying egg in February, the male remains in a riding position upon the female for approximately 24 days. Under artificial short-day conditions the riding position may also be assumed outside of the reproductive period. The eggs are transported to the ovigers of the male by trunk movements of both partners. At 12°C the larvae hatch about 41–46 days after egg-laying. No larvae hatched from eggs laid during summer at 19°C.The larvae live endoparasitically in Hydrozoa for about 1/2 year. Following metamorphosis, the freeliving juvenile phase lasts barely a year. The maturation moult normally takes place in the summer of the second year, the reproductive period beginning about 6 months later, in winter.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.     

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.     

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

The Influence of Hunting on Antipredator Behavior in Central African Monkeys and Duikers

Many animals can adjust their behavioral strategies to reduce predation risk. We investigated whether rain forest monkeys and duikers alter their antipredatory behavior in response to hunting by humans in southwestern Gabon. We compared monkey and duiker responses to human observers in an area where hunting is prohibited, to those in a nearby area where hunting pressure is moderate but spatially variable. The results of our study indicate that monkeys become more secretive when hunted, commencing alarm calls only when at a certain distance (typically > 50 m) from humans. We found no difference in monkey group size between hunted and no-hunting areas. In no-hunting areas, duikers often freeze in response to approaching observers, but in hunted areas they abandon this strategy and rapidly flee from humans. Duikers also whistle more often in areas where they are hunted frequently. Our findings have at least two important implications. First, behavioral observations of monkeys and duikers may be useful in gauging local hunting intensity in African rain forests. Second, duiker densities are likely to be overestimated in hunted areas, where they more readily flee and whistle, and underestimated in no-hunting areas, where they rely on freezing behavior to avoid detection. Because behavioral adaptations to hunting vary both among species and localities, these differences should be considered when attempting to derive population-density estimates for forest wildlife.