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G D'Alessio

Publications and source records attributed to G D'Alessio.

11 recordsLinked to original sources
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↗
PubMed1975

Degradation of double-stranded RNA by a monomeric derivative of ribonuclease BS-1.

Double-stranded RNA, resistant to the action of pancreatic monomeric RNAase A, is actively degraded by seminal dimeric RNAase BS-1. Evidence is presented that a monomeric derivative of seminal RNAase degrades double-stranded RNA as efficiently as the parent dimeric molecule. This finding is discussed in the light of the hypothesis previously advanced that two active sites simultaneously available on an enzyme molecule may be responsible for degradation of double-stranded polyribonucleotides.

Animals↗
PubMed1975

Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.

The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.

Alkylation↗
PubMedDate not supplied

Monomeric selectively S-alkylated derivatives of seminal ribonuclease: preparation and properties.

Procedures are described for preparing monomeric selectively S-carboxamido-methylated and S-aminoethylated derivatives of seminal ribonuclease. The main properties of the derivatives, including their extinction coefficients, have been determined. Their catalytic activities and that of the S-carboxymethyl derivative have been tested. On double-stranded RNA as a substrate the monomeric derivatives are less active than the native dimeric enzyme, but much more active than pancreatic ribonuclease. On yeast RNA as a substrate the amino-ethyl derivative is found to be less active (80%) than the native enzyme, while the other two are over 30 percent more active. The monomers are stable in solution and when lyophilized from acetic acid solution do not associate to the same extent as pancreatic or native seminal ribonucleases.

Alkylation↗