Biomedical subjects

M Tamburrini

Publications and source records attributed to M Tamburrini.

25 records · Page 2Linked to original sources

PubMed1991

Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide.

Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G "transamidase" derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2- and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted beta carbon at the cleavage/attachment site that were found previously with wild type PLAP.

Acyltransferases↗

PubMed1991

Oxygen transport in extreme environments.

Evolution has adopted different strategies to solve the problem of transporting oxygen to respiring tissues, according to needs dictated by the environment. A thermodynamic analysis of haemoglobins of organisms living in extreme polar environments (mammals and fish) provides elegant examples of such adaptations.

Adaptation, Physiological↗

PubMed1990

Immunosuppressive activity of bovine seminal RNase on T-cell proliferation.

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.

CD3 Complex↗

PubMed1989

Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.

Amino Acids↗

PubMed1987

Autocrine growth function of human interleukin 1 molecules on ROHA-9, an EBV-transformed human B cell line.

In this study we report that the ROHA-9 cell line, an IL 1-secreting EBV-transformed human B cell line, exhibits an autocrine pathway of growth. In fact, ROHA-9 cells spontaneously secreted an autoregulatory growth factor that co-purified with the constitutively secreted IL 1-like molecules. Accordingly, monocyte-derived human IL 1, free of other known biological activities, also stimulated the growth of ROHA-9 cells in a dose-dependent way. Human recombinant interleukin 2, recombinant IFN-alpha or IFN-gamma and purified IFN-beta were ineffective when used at concentrations up to 1 X 10(3) U/ml. Furthermore, mouse recombinant IL 1, HPLC-purified multi-colony stimulating factor and partially purified preparations of BCGF were ineffective when assayed for growth-promoting activity on ROHA-9 cells. Moreover, a rabbit polyclonal antibody and a mouse monoclonal antibody to human IL 1 molecules blocked the growth of ROHA-9 cells induced by the autologous growth factor and by human IL 1. Lastly, purified human IL 1 increased the clonal efficiency of ROHA-9 cells seeded at a low cell concentration, allowing the isolation of the ROHA-9MC3 subclone, which showed similar growth response specificity and was particularly sensitive to the mitogenic activity of human IL 1.

Animals↗

PubMed1986

Site-directed alkylation and site-site interactions in bovine seminal ribonuclease.

Active-site histidine residues of bovine seminal RNase have been found to react with bromoacetic acid and with 2'(3')-O-bromoacetyluridine (BrAcUrd) at a much faster rate than free histidine. The former reagent reacts preferentially at the pros-N of His119, the latter is specific for the tele-N of His12. Alkylation with bromoacetic acid is mutually exclusive for either His119 or His12 and takes place predominantly at His119, while with BrAcUrd alkylation was found to be selective for His12. These results are very similar to those obtained with the same reagents on RNase A, confirming that seminal and pancreatic ribonucleases have similar geometries at their active sites. On the other hand, the kinetics of reaction of bromoacetyluridine with seminal RNase reveal a 'half-of-the sites' reactivity of the enzyme for this reagent, which is found to discriminate between the two structurally identical active sites of the dimeric enzyme.

Acetates↗

PubMedDate not supplied

Fast and high-yielding procedures for the isolation of bovine seminal RNAase.

Fast and high yielding procedures for the isolation of bovine seminal RNAase are described. Homogeneous enzyme is prepared from seminal plasma in high yields in a single chromatographic step. Higher amounts (hundreds of mg) are easily prepared from seminal vesicles, a more available source of enzyme. Both procedures can be used also for the direct isolation of the isoenzymes of bovine seminal RNAase. An ultrarapid (1 hour) procedure is described for the preparation of mg amounts of pure enzyme, or of the individual isoenzymes, from seminal plasma.

Animals↗