The SEA module: a new extracellular domain associated with O-glycosylation.

Using a variety of homology search methods and multiple alignments, a new extracellular module was identified in (1) agrin, (2) enterokinase, (3) a 63-kDa sea urchin sperm protein, (4) perlecan, (5) the breast cancer marker MUCI (episialin), (6) the cell surface antigen 114/A10, and (7/8) two functionally uncharacterized, probably extracellular, Caenorhabditis elegans proteins. Despite the functional diversity of these adhesive proteins, a common denominator seems to be their existence in heavily glycosylated environments. In addition, the better characterized proteins mentioned above contain all O-glycosidic-linked carbohydrates such as heparan sulfate that contribute considerably to their molecular masses. The common module might regulate or assist binding to neighboring carbohydrate moieties.     

Exhaustive enumeration of protein conformations using experimental restraints.

We present an efficient new algorithm that enumerates all possible conformations of a protein that satisfy a given set of distance restraints. Rapid growth of all possible self-avoiding conformations on the diamond lattice provides construction of alpha-carbon representations of a protein fold. We investigated the dependence of the number of conformations on pairwise distance restraints for the proteins crambin, pancreatic trypsin inhibitor, and ubiquitin. Knowledge of between one and two contacts per monomer is shown to be sufficient to restrict the number of candidate structures to approximately 1,000 conformations. Pairwise RMS deviations of atomic position comparisons between pairs of these 1,000 structures revealed that these conformations can be grouped into about 25 families of structures. These results suggest a new approach to assessing alternative protein folds given a very limited number of distance restraints. Such restraints are available from several experimental techniques such as NMR, NOESY, energy transfer fluorescence spectroscopy, and crosslinking experiments. This work focuses on exhaustive enumeration of protein structures with emphasis on the possible use of NOESY-determined distance restraints.     

Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.     

Structure of the histone tetramer (H3-H4)2: 2. Position of λmax in the tyrosyl fluorescence spectra and tyrosyl accessibility to quenchers

It was shown that the histone tetramer (H3-H4)₂ fluorescence spectra were shifted by about 2 nm towards the long-wave region and had a larger halfwidth than the free tyrosine fluorescence spectra. Denaturation with 8 m urea resulted in a shift towards the short-wave region and a decrease in the halfwidth of the histone tetramer (H3-H4)₂ tyrosine fluorescence spectra. Fluorescence quenching of the histone tetramer (H3-H4)₂ by iodine ions was analysed by the Stern-Volmer equation. It was estimated that at 0.1 m NaCl and 0.3–0.8 m NaCl, 45% and 60% tyrosyl fluorescence, respectively, was quenched by I^? ions. The results obtained suggests that histone tetramer (H3-H4)₂ may have several structural forms distinguished by the amount of ‘exposed’ and ‘buried’ tyrosyls depending upon the conditions of the medium.     

Salt-tolerance in plants. I. Ions, compatible organic solutes and the stability of plant ribosomes

Abstract Polysomes and ribosomes recovered from a number of plant species were tested for stability when incubated at 25°C in salt solutions in the absence of ATP and initiation factors. Stability was assessed by sucrose density gradient analysis. The stability was inversely proportional to salt concentrations above 125 mol m⁻³ KCl. Polysomes were less stable in the presence of Na⁺ than K⁺ salts, and were much less stable in Cl⁻ than in acetate salts. Polysomes from Triticum aestivum. Hordeum vulgare, Capsicum annuum, Helianthus annuus. Pisum sativum, Atriplex nummularia, Beta vulgaris, Cladophora sp., Enteromorpha sp. and Corallina cuvieri were similarly sensitive to KCl. Polysomes from Ulva lactuca were more sensitive than the other species. Cytoplasmic and plastid polysomes from T. aestivum were similarly unstable in 500 mol m⁻³ KCl. Unprogrammed ribosomal subunit couples from T. aestivum, B. vulgaris and U. lactuca showed Mg²⁺-dependent conformational instability and dissociation in KCl. Slight differences in ribosomal stability were observed between species, but these were unrelated to the salt tolerances of the plants. The ‘compatible’ organic solutes, glycinebetaine and proline, failed to reduce ion-induced instability. Ribosome yield and polysome profiles were similar in leaves of B. vulgaris containing significantly different levels of both Na⁺ and Cl⁻ after growth in media containing 50 or 200 mol m⁻³ NaCl. The results are consistent with the hypothesis that plants maintain a cytoplasmic solute environment that is compatible with ribosomal stability.     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.     

A spin-labeled derivative of Procion brilliant blue MX-R. Synthesis and interaction with pig heart nucleoside diphosphate kinase

The ¹H-NMR spectrum of cucumber basic blue protein (CBP) has been recorded. Examination of the spectrum of the reduced protein suggests that one or more sidechains exist in conformations which interconvert slowly at ambient temperatures. His 39, His 84 and Met 89 are identified as copper ligands by redox titration and by amino acid sequence homology with plastocyanin and azurin. The importance of a Phe sidechain close to the Met ligand in the potential blue copper site is confirmed. Broadening of His ligand resonances at elevated temperatures reveals an exchange process at the reduced copper centre.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Changes in intracellular proteolysis in Escherichia coli during prolonged growth with a low concentration of dihydrostreptomycin

Abstract Escherichia coli was cultured with a low concentration of dihydrostreptomycin (2.5 μg/ml.). Growth was similar to untreated controls for 10 h after which a slow decline in growth rate occurred; growth ceased after 20 h. Intracellular catabolism of pulse-labelled protein synthesised at various points during the antibiotic treatment increased during the first 10 h, but during the second 10 h proteolysis progressively declined to almost control values. The production of an aberrant proteolytic system is one possible explanation.     

Contribution to the structural characterization of eucaryotic PSI reaction centre—I. Critical analysis of the polypeptidic composition of different P700 enriched fractions

In thylakoid membranes, several peptides of high MW are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The and subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.