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M Libonati

Publications and source records attributed to M Libonati.

At least 19 recordsLinked to original sources
PubMed2001

Different susceptibility of the two dimers of ribonuclease A to subtilisin. Implications for their structure.

RNase A and its minor and major dimers were digested with subtilisin under controlled conditions. The major dimer was found to be slightly more resistant, the minor dimer markedly less resistant to subtilisin than monomeric RNase A. Two S-proteins formed for each RNase A species, one starting with Ser-21, the other with Ser-22. Their relative proportions indicate that the structure of the minor dimer, whose identity with that of a RNase A dimer shown to be 3D domain-swapped is strongly suggested by recent work [S. Sorrentino et al. (2000) FEBS Lett. 466, 35-39], makes its peptide bond between Ser-21 and Ser-22 more accessible to subtilisin than it is in RNase A and its major dimer. Moreover, (i) both subunits constituting the minor dimer are more susceptible to subtilisin than monomeric RNase A, and (ii) the susceptible bonds in one of its two exchanging N-terminal arms are more accessible to the protease than in the other. The properties of the major dimer suggest that its structure could be different.

Animals↗
PubMed2001

A domain-swapped RNase A dimer with implications for amyloid formation.

Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal alpha-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal beta-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped beta-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.

Animals↗
PubMed2001

Structural properties of trimers and tetramers of ribonuclease A.

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.

Animals↗
PubMed2000

The two dimeric forms of RNase A.

In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868-3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X-ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437-3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A.

Animals↗
PubMed1999

Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Amino Acid Sequence↗
PubMed1999

Structural versatility of bovine ribonuclease A. Distinct conformers of trimeric and tetrameric aggregates of the enzyme.

Lyophilization of bovine ribonuclease A (RNase A; Sigma, type XII-A) from 40% acetic acid solutions leads to the formation of approximately 14 aggregated species that can be separated by ion-exchange chromatography. Several aggregates were identified, including two variously deamidated dimeric subspecies, two distinct trimeric and two distinct tetrameric RNase A conformers, besides the two forms of dimer characterized previously [Gotte, G. & Libonati, M. (1998) Two different forms of aggregated dimers of ribonuclease A. Biochim. Biophys. Acta 1386, 106-112]. We also have possible evidence for the existence of two forms of pentameric RNase A. The two forms of trimers and tetramers are characterized by: (a) slightly different gel filtration patterns; (b) different retention times in ion-exchange chromatography; and (c) different mobilities in cathodic gel electrophoresis under nondenaturing conditions. Therefore, they appear to have distinct structural organizations responsible for a different availability of their positively charged amino acid residues. All RNase A oligomers, in particular the two distinct trimeric and tetrameric conformers, degrade poly(A).poly(U), viral double-stranded RNA and polyadenylate with a catalytic efficiency that is in general higher for the more basic species. On the contrary, the activity of the RNase A oligomers, from dimer to pentamer, on yeast RNA and poly(C) (Kunitz assay) is lower than that of monomeric RNase A.

Animals↗
PubMed1998

Two different forms of aggregated dimers of ribonuclease A.

Results of gel filtration experiments performed with two different chromatographic media (Superose 12 HR 10/30 and Superdex 75 HR 10/30) and of polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions suggest that aggregated dimers of bovine RNase A, obtained by lyophilization of the enzyme from 40% acetic acid solutions (5 mg RNase A per ml), might consist of two differently structured forms. These two species have slightly different retention times in gel-filtration experiments and migrate differently in electrophoresis under non-denaturing conditions. The fast migrating dimer in non-denaturing gel electrophoresis is able to degrade double-stranded poly(A).poly(U) more efficiently than the other, and the two forms are found in a ratio of about 3:1.

Chromatography, Gel↗
PubMed1998

Lithostathine messenger RNA expression in different types of chronic pancreatitis.

Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous tissue from pancreatic cancer subjects. However, about 70% of the pancreatic cancer subjects showed lithostathine and chymotrypsinogen mRNA levels comparable to those of chronic pancreatitis patients. These results indicate that the decrease in the level of mRNA is not specific to lithostathine and it is unrelated to the presence of pancreatic stones.

Adult↗
PubMed1997

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action.

Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.

Animals↗
PubMed1997

Structure-function relationships in human ribonucleases: main distinctive features of the major RNase types.

Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase superfamily, can be classified into four different enzyme types on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and catalytic differences (action on single- and double-stranded RNAs, dependence of enzyme activity on pH, ionic strength and cations, and hydrolysis of cyclic nucleotides) have been comparatively analyzed and discussed. In addition, some data considered here concerning human nonpancreatic-type RNases may support the suggestion [Chuchillo et al. (1993) FEBS Lett. 333, 207-210] that the enzyme 'ribonuclease', presently classified as 'hydrolase', should be reclassified as 'transferase'.

Amino Acid Sequence↗
PubMed1997

Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson's disease.

Current approaches to gene therapy of CNS disorders include grafting genetically modified autologous cells or introducing genetic material into cells in situ using a variety of viral or synthetic vectors to produce and deliver therapeutic substances to specific sites within the brain. Here we discuss issues related to the application of ex-vivo and in-vivo gene therapies as possible treatments for Parkinson's disease. Autologous monkey fibroblasts engineered ex-vivo to express tyrosine hydroxylase were grafted into MPTP-treated monkeys and found to express for up to 4 months. Adeno-associated (AAV) viral vectors expressing beta-galactosidase or tyrosine hydroxylase were introduced into monkey brains to determine the extent of infection and the types of cells infected by the vector at 21 days and 3 months. Gene expression was detected at both time points and was restricted to neurons in the striatum. These experiments demonstrate that two different approaches can be used to deliver proteins into the CNS. However, further technological advances are required to optimize gene delivery, regulation of gene expression, and testing in appropriate functional models before gene therapy can be considered for treating human disease.

Animals↗
PubMed1997

Single-strand-preferring RNases degrade double-stranded RNAs by destabilizing its secondary structure.

To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.

Animals↗
PubMed1996

The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates.

Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40-50% acetic acid solutions. The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides. Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates. In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer. These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.

Animals↗
PubMed1996

Selective association of a 22-38 kDa glycoprotein with MHC class II DP antigen on activated human lymphocytes at the plasma membrane.

Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.

B-Lymphocytes↗
PubMed1995

Double-stranded RNA: the variables controlling its degradation by RNases.

The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.

Animals↗
PubMed1994

Human pancreatic-type and nonpancreatic-type ribonucleases: a direct side-by-side comparison of their catalytic properties.

The catalytic properties and substrate preference of several highly purified human ribonucleases from different organs and body fluids have been examined in detail using various low-molecular-weight compounds and single- or double-stranded polyribonucleotides as substrates. All single-stranded polyribonucleotides were degraded by nonpancreatic-type (npt) RNases at a slower rate than by pancreatic-type (pt) enzymes: ptRNases were 20 times more active on RNA and poly(U) substrates and more than 6000 times more active on poly(C). Pancreatic-type RNases degraded poly(C) faster than RNA, showing a strong preference for poly(C) over poly(U) with the following activity ratios: RNA/poly(C), 0.44; RNA/poly(U), 12; poly(C)/poly(U), 27. In contrast, nptRNases cleaved RNA more rapidly than synthetic homopolymers, preferring poly(U) over poly(C) with the following ratios: RNA/poly(C), 130; RNA/poly(U), 10; poly(C)/poly(U), 0.08. Human ptRNases degraded poly(A) and double-stranded polyribonucleotides about 100 and 400 times faster, respectively, than bovine RNase A. However, no measurable activity could be detected on these substrates with nptRNases. The activities of ptRNases on dinucleoside phosphates (CpN and UpN) or uridine and cytidine 2',3'-cyclic phosphates were similar to those of bovine RNase A; nptRNases, instead, cleaved only CpA and UpA at an appreciable rate. The effects of pH, ionic strength, and divalent cations on the activity of these ribonucleases were also investigated using yeast RNA as a substrate.

Catalysis↗
PubMed1993

Differential effects of tyrosine kinase inhibition in CD69 antigen expression and lytic activity induced by rIL-2, rIL-12, and rIFN-alpha in human NK cells.

The effect of rIL-12 on induction of CD69 antigen expression and cytolytic activity in purified human NK cells was evaluated in comparison to the effects of rIL-2 and rIFN-alpha. It was found that rIL-12 directly induced CD69 antigen expression in NK cells, although the period of incubation required by rIL-12 was longer than the period required by rIL-2 or by rIFN-alpha. Similarly, the cytolytic activity induced by rIL-12 in NK cells against the NK-resistant target cell line Raji was consistently lower than the cytolytic activity induced by rIL-2 or rIFN-alpha when measured after 6 hr of incubation, and increased during the following 18 hr of incubation. To compare the involvement of tyrosin kinases in activation of NK cells induced by rIL-2, rIL-12, and rIFN-alpha, the effect of the specific inhibitor of tyrosin kinases, genistein, was evaluated on induction of CD69 antigen expression and lytic function mediated by the three cytokines. It was found that genistein inhibited CD69 antigen expression induced by rIL-2 and by rIL-12, but not that induced by rIFN-alpha. Unlike the effect on CD69 antigen expression, the cytolytic activity induced by all three cytokines was inhibited by genistein. These results, together with the finding that CD69 antigen expression induced by rIL-2 but not by rIL-12 or rIFN-alpha was inhibited by addition of rIL-4, strongly suggest that IL-2, IL-12, and IFN-alpha mediate their effects, leading to induction of CD69 antigen expression through different activation pathways.

Antigens, CD↗