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Biomedical subjects

G D'Alessio

Publications and source records attributed to G D'Alessio.

At least 73 records · Page 4Linked to original sources
PubMed1985

Reacquisition of quaternary structure by fully reduced and denatured seminal ribonuclease.

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.

Air↗
PubMed1985

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease.

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.

Amino Acids↗
PubMed1984

Relationships between nonhyperbolic kinetics and dimeric structure in ribonucleases.

Dimers of bovine pancreatic RNase A give nonhyperbolic saturation curves for the substrate of the second, rate-limiting step of the reaction. Under the same conditions, the monomeric native enzyme shows Michaelis-Menten kinetics. Naturally dimeric bovine seminal RNase, which has been found to give nonhyperbolic saturation curves, loses this property upon monomerization. It is proposed that when RNase monomers are arranged in a quaternary structure, they assume a conformation which enables them to be modulated in their catalytic activities. A correlation is suggested between this effect and the quaternary structure proposed for both of these dimeric ribonucleases, in which composite active sites are generated by the mutual exchange of the NH2-terminal ends of the two monomers.

Animals↗
PubMed1983

A fucosyl glycoprotein component with sperm receptor and sperm-activating activities from the vitelline coat of Ciona intestinalis eggs.

In this paper we describe a mild procedure which results in the extraction of a glycoprotein fraction from the vitelline coat (VC) of Ciona intestinalis while leaving behind the bulk of the VC components. When acting upon the spermatozoa this fraction inhibits sperm binding to the VC and fertilization and elicits sperm activation including the acrosome reaction. SDS-PAGE shows that it contains the same (fucosyl) glycoprotein components previously recognized in the total extracts of VC. It is suggested that this material contains the sperm receptors or those components of the receptors that are essential for their Chinese function.

Animals↗
PubMed1983

Comparative proton NMR studies of bovine semen and pancreas ribonucleases.

The fine structure of bovine semen RNAase was studied with proton NMR spectroscopy making use of the four-protein system constituted by dimeric bovine semen RNAase, its catalytically active monomeric bis-(S-carboxymethyl-31,32) derivative, the naturally monomeric RNAase A from the pancrease of the same species, and dimerized RNAase A. Only four histidine C-2 H resonances were observed in the aromatic spectrum of bovine semen RNAase, which belong to the four histidine residues present in the sequence of bovine semen RNAase subunits at positions identical with those of the histidines of RNAase A. This is indicative of identical environments for the individual histidine residues in both subunits. These resonances were assigned (i) by comparing their titration curves with the corresponding curves obtained with RNAase A and with monomeric bovine semen RNAase and (ii) by evaluating the effects on their titration curves of nucleotide binding. Very similar NMR parameters were measured for His-105 and also for His-119 of seminal and pancreatic RNAase, while His-12 was found to have different environments in the two proteins. The distinctive NMR features of His-48 in bovine semen RNAase confirmed the role of the hinge regions of the subunits in maintaining the dimeric structure of the protein. While monomerization of the seminal enzyme reduced the differences between the histidine C-2 H resonances of RNAase A and bovine semen RNAase, dimerization of RNAase A did not affect the NMR spectrum of this protein, thus indicating as unlikely the possibility that the quaternary structure of bovine semen RNAase resembles that of dimerized RNAase A.

Animals↗
PubMed1981

Heterogeneity of bovine seminal ribonuclease.

Bovine seminal ribonuclease, a dimeric protein found to be homogeneous by several standard criteria of purity, is heterogeneous when analyzed by ion-exchange chromatography on (carboxymethyl)cellulose. Three increasingly cationic subforms can be separated. The heterogeneity is due to the presence of two types of subunits, alpha and beta, which make up three isoenzymic dimers: alpha 2, beta 2, and alpha beta. Deamidation reactions can convert the most cationic beta 2 subform into the alpha beta subform, which in turn can be converted into stable alpha 2 subform. These conversions involve the hydrolysis of 2 mol of differentially labile amide groups per mol of protein. The ratios alpha 2: alpha beta: beta 2 are constant in all preparations of seminal ribonuclease tested; they are independent of the purification procedure as well as of the biological source of the enzyme (seminal plasma or seminal vesicles). These results indicate that deamidations occur in vivo before the protein is secreted from the seminal glands. They also suggest that heterogeneity of seminal ribonuclease reflects a physiological need of distinct molecular forms of enzyme or, alternatively, a process which leads to the aging of the protein.

Animals↗
PubMed1980

Transport and fate of gaseous pollutants associated with the National Energy Program.

The experimental evidence related to the long-range transport and transformations of sulfates has been critically reviewed. It has been concluded that sulfate emissions from various sources can be superimposed on each other during long-range transport to create episodes far from the contributing sources. Sulfates are important contributors to degrading visibility through the United States including relatively pristine areas in the western United States. Acid sulfate components, along with nitric acid, may increase the problem of acidity in precipitation on soils, vegetation, and lakes in certain areas of the eastern United States. Improvements in monitoring of these pollutants are required to follow changes in air quality caused by long-range transport.

Air Pollutants↗
PubMed1980

In vitro studies on selective inhibition of tumor cell growth by seminal ribonuclease.

The effect on cell growth of bovine seminal RNase has been tested on cells cultured in vitro. A selective inhibition of growth has been observed on tumor cells as compared to normal cells using several virus-transformed cell lines and a neuroblastoma line. The different cellular response of virus-transformed cells does not appear to depend on a differential permeability to the protein of transformed cells with respect to nontransformed ones. The selective cytotoxic action of seminal RNase on tumor cell growth was also compared with the action of other structurally related RNases and of various RNase derivatives prepared by specific chemical modifications. The results indicate that the protein dimeric structure and its enzymic activity are essential requirements for its action.

Animals↗
PubMed1979

Intrachain disulfide bridges of bovine seminal ribonuclease.

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.

Amino Acid Sequence↗
PubMed1978

Glutathione-facilitated refolding of reduced, denatured bovine seminal ribonuclease: kinetics and characterization of products.

Totally reduced and denatured seminal ribonuclease was regenerated using the glutathione redox system. The refolding kinetics of the enzyme were determined as a function of redox state, temperature from 14 to 43 degrees C, pH, and protein concentration. The maximal rate of regeneration occurred with 3 x 10(-3) M reduced glutathione, 6 x 10(-4) M oxidized glutathione, 24 to 30 degrees C, and pH 8.2. The products of the refolding process were characterized by Sephadex G-75, sodium dodecyl sulfate gel electrophoresis, enzymatic activity, circular dichroism, and amino acid analysis. The results indicate that the native dimeric form of the enzyme is not produced during refolding to any appreciable extent; rather, the major product is monomeric. The purified monomer exhibits twice the activity of the native enzyme toward yeast RNA. Its circular dichroism spectrum is different from the native enzyme and is quite similar to that of pancreatic ribonuclease A. Amino acid analyses showed that two glutathione molecules are bound to the monomer, suggesting that cysteine-31 and -32, which normally form the intermolecular disulfide bonds, are blocked.

Amino Acids↗
PubMed1976

Proteolytic enzymes as structural probes for ribonuclease BS-1.

Trypsin, pepsin and subtilisin have been used as conformational probes for the structure of bovine seminal ribonuclease BS-1 by studying, under definite conditions, their effects on the seminal enzyme, a dimeric protein made up to two identical subunits; on bovine pancreatic monomeric ribonuclease A (EC 3.1.4.22) with a polypeptide chain homologous to that of the seminal ribonuclease subunit chain; and on a monomeric, active and stable derivative of seminal ribonuclease. The results show: (1) that the C-terminal regions of the pancreatic and the seminal proteins are very similar as they appear to fit in an identical way to the active site of pepsin; (2) that the resistance of the N-terminal region of ribonuclease BS-1 to subtilisin is not due to the dimeric structure of the protein, but to the conformation of this region, where an essential feature is the presence of a proline residue at position 19; (3) that the monomer of ribonuclease BS-1 is resistant to tryptic action only when bound to the partner monomer in the quaternary structure of the protein. This indicates that dissociation of the seminal ribonuclease makes some potentially susceptible susceptible bond or bonds available to trypsin either through a conformational change of the protein subunit, or by simply exposing the protein area hidden at the intersubunit interfaces.

Animals↗