Biomedical subjects
Publications and source records attributed to A Parente.
The K-FAST and K-SNAP, two new brief cognitive measures designed for adolescents and adults, were validated against another brief measure--a four-subtest short form of the WAIS-R--using a sample of 20 adult patients hospitalized for depression. Data supported the validity of these two new instruments.
Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkin's lymphoma-derived L540 cell line.
A lectin was purified from the latex of Euphorbia marginata by affinity chromatography on acid-treated Sepharose 6B and elution with lactose. The lectin is a glycoprotein composed of two identical subunits with M(r) 30,000, approx. The haemagglutinating activity of the lectin is not specific for any human blood group, and is inhibited by galactose and galactose-containing sugars and by gentiobiose. The lectin is strongly mitogenic for human T-lymphocytes and induces the release of interleukin-1 beta and tumor necrosis factor-alpha from cultured mononuclear cells.
The complete amino-acid sequence of the dimeric and cooperative myoglobin from the radular muscles of Nassa mutabilis, a common edible gastropod mollusc on the Italian coast, has been determined. The molecule is a homodimer. The monomer is composed of 147 amino-acid residues, with a molecular mass of 15,760 Da. Its sequence is homologous with those of the dimeric myoglobins of the gastropod molluscs of the Prosobranchia subclass Busycon canaliculatum (63% conserved residues) and Cerithidea rhizophorarum (46% conserved residues). The rate of autoxidation to met-myoglobin of N. mutabilis oxymyoglobin at 25 degrees C is strongly pH-dependent with relative minimal rate values in the pH range 7 to 8.
The elongation factor 2 (aEF-2) from the extreme thermo-acidophilic archaebacterium Sulfolobus solfataricus, is the only cytosolic target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD. Once ADP-ribosylated, aEF-2 is no longer able to sustain poly(Phe) synthesis in vitro. aEF-2 displays a great thermoresistance: at the growth temperature of the archaebacterium, 87 degrees C, its half-life is 3 h. The amino acid sequence of the N-terminal region of aEF-2 has been determined up to residue 22. In the first 15 positions such a sequence is identical to that of EF-2 from Sulfolobus acidocaldarius and very similar to that of EF-2 from other archaebacteria or eukaryotes. The same is true for the primary structure of the peptide containing the ADP-ribosylation site. The fact that the primary structure of EF-2 at the ADP-ribosylation site is highly conserved ensures either the correct recognition of the histidine residue by the enzymes involved in its modification to diphthamide, or the proper interaction with the diphtheria toxin.
Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.
The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)
The RNase found in bull semen, although a member of the mammalian superfamily of ribonucleases, possesses some unusual properties. Besides its unique structure and enzymic properties, it displays antispermatogenic, antitumor and immunosuppressive activities. Seminal RNase belongs to an interesting group of RNases, the RISBASES (RIbonucleases with Special, i.e. non catalytic, Biological Actions) other members of which include angiogenin, selectively neurotoxic RNases, a lectin and the self-incompatibility factors from a flowering plant.
The interest in the immunosuppressive activity of mammalian seminal plasma depends largely on its putative role in the immunoregulation of both the male and female genital systems. We report here that the immunosuppressive action of bovine seminal plasma is based on the presence in this fluid of copious amounts of an immunosuppressive RNase, bovine seminal RNase. Studies of structure-function relationships have revealed that the immunosuppressive activity of seminal RNase depends on the integrity of the dimeric structure of the enzyme, as well as on the integrity of its catalytic function. While bovine seminal RNase has no effect on the secretion of interleukin-2 by T-cell cultures, the enzyme has been found to decrease drastically the expression of the alpha-chain of the interleukin-2 receptor on the T-cell membrane.
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We have isolated a new toxin, calitoxin (CLX), from the sea anemone Calliactis parasitica whose amino acid sequence differs greatly from that of other sea anemone toxins. The polypeptide chain contains 46 amino acid residues, with a molecular mass of 4886 Da and an isoelectric point at pH 5.4. The amino acid sequence determined by Edman degradation of the reduced, S-carboxymethylated polypeptide chain and tryptic and chymotryptic peptides is Ile-Glu-Cys-Lys-Cys-Glu-Gly-Asp-Ala-Pro-Asp-Leu-Ser-His-Met-Thr-Gly-Thr- Val-Tyr - Phe-Ser-Cys-Lys-Gly-Gly-Asp-Gly-Ser-Trp-Ser-Lys-Cys-Asn-Thr-Tyr-Thr-Ala- Val-Ala - Asp-Cys-Cys-His-Glu-Ala. No cysteine residues were present in the peptide. Similarly to other sea anemone toxins, calitoxin interacts, in crustacean nerve muscle preparations, with axonal and not with muscle membranes, inducing a massive release of neurotransmitter that causes a strong muscle contraction. The low homology of CLX with RP II and ATX II toxins has implications regarding the role played by particular amino acid residues.
The presence of an acetyl blocking group at the N-terminus of the coat protein of papaya mosaic virus has been identified by FAB mass spectrometry. Furthermore, we have found that the N-terminal sequence of the protein is four amino-acid residues (AC-Ser-Lys-Ser-Ser-) longer than that previously reported, while Glu instead of Gln is the C-terminal residue. The present paper shows that PMV-protein is made up of 215 amino acid residues, with a molecular mass of 22,960 Da.
1. Adult chicken hemoglobins were analysed by ion exchange chromatography and isoelectric focusing and a minor hemoglobin fraction (HbK) was isolated. 2. Analysis of the constituent chains shows that HbK differs from the two major hemoglobins HbA and HbD in the alpha globin. 3. The amino acid composition, the tryptic peptide maps, the results of carboxypeptidase digestion and the functional properties show that the HbK alpha globin is quite similar to that of HbA except that the C-terminal amino acid Arg 141 is lacking. 4. HbK must then be considered a Koelliker-type hemoglobin.
The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.
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Air-regenerated monomers of bovine seminal ribonuclease have been found capable of reassociating into native dimers, whereas monomers refolded in the presence of a glutathione redox mixture do not reassociate into dimers [Smith, K. G., D'Alessio, G. and Schaffer, S. W. (1978) Biochemistry 17, 2633-2638]. The crucial step in the process of regeneration of dimers is an isomerization step, which the newly refolded monomers undergo in order to reassociate into dimers. The two sulfhydryls at sequence positions 31 and 32 of the seminal RNAase chain, forming in the native dimer the intersubunit disulfides, have been found to have an important role in the refolding of the monomeric intermediates, as well as in the regeneration of dimers.
The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.
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