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Biomedical subjects

L Calabrese

Publications and source records attributed to L Calabrese.

At least 109 records · Page 6Linked to original sources
PubMed1983

Sheep ceruloplasmin: isolation and characterization.

Ceruloplasmin has been isolated from sheep plasma by a procedure involving two chromatographic steps and (NH4)2SO4 fractionation. The ovine protein is similar to ceruloplasmins from other species previously described (human, bovine), having a single chain of about 125 Kdal with a very high degree of homology in the amino acid composition. It differs, however, from human and bovine ceruloplasmin because of its lower copper content and its higher specific enzyme activity. The oxidase activity as well as the spectroscopic properties were found to be pH range 5-8 with a pH optimum for activity of 6.3.

Amino Acids↗
PubMed1982

Synthesis of either Fe- or Mn-superoxide dismutase with an apparently identical protein moiety by an anaerobic bacterium dependent on the metal supplied.

Superoxide dismutase of Propionibacterium shermanii, an anaerobic that produces an iron superoxide dismutase, was purified from cells grown in iron-free conditions. The enzyme isolated was found to contain manganese and to have spectral and catalytic properties very similar to those of typical Mn-superoxide dismutases. Its electrophoretic mobility, molecular weight, and subunit size were identical with those of the Fe-enzyme. Amino acid compositions were practically indistinguishable in either case. The NH2-terminal sequence was found to be identical. The catalytic activity of an apoprotein sample prepared from the purified holoenzyme was restored by adding either Mn(II) or Fe(II). Only the metal/protein ratio varied from approximately 1 per subunit in the case of the Fe-enzyme to approximately 2 for the Mn-enzyme. It is concluded that this bacterium can accommodate either Fe or Mn on identical, or very slightly dissimilar, proteins forming active sites with the properties found in specific metallodismutases.

Amino Acids↗
PubMed1982

Isolation and preliminary characterization of electrophoretic variants of copper, zinc superoxide dismutase.

Three electrophoretic variants of superoxide dismutase can be detected in bovine erythrocytes by gel electrophoresis and electrofocusing. The two major forms, having isoelectric points at pH 5.2 and 4.9, were isolated by preparative focusing or chromatography. No differences were found in molecular weight, metal content, antigenicity, electron spin resonance spectrum, visible and ultraviolet optical spectra. In contrast, holo- and apo-superoxide dismutase, which have an electrophoretic mobility similar to that of the two major forms, showed unresolved isoelectric points but significantly different antigenicity. This result suggests that their different electrophoretic mobility is mainly conformation-related. The variant with pI 5.2, corresponding to the protein purified by ordinary procedures, was found to be inactivated by heat treatment faster than the other form. The latter one, on the other hand, gave rise to a multiple pattern of electrophoretic bands after incubation at 75 degrees C. It is suggested that superoxide dismutase multiplicity in erythrocytes is not genetically determined, but may be related to segregation of subunits, made non-identically by post translational asymmetrical modification.

Animals↗
PubMed1982

Evidence for catalytic dismutation of superoxide by cobalt(II) derivatives of bovine superoxide dismutase in aqueous solution as studied by pulse radiolysis.

By using the technique of pulse radiolysis to generate O2-., it is demonstrated that Co(II) derivatives of bovine superoxide dismutase in which the copper alone and both the copper and zinc of the enzyme have been substituted by Co(II), resulting in (Co,Zn)- and (Co,Co)-proteins, are capable of catalytically dismutating O2-. with 'turnover' rate constants of 4.8 X 10(6) dm3.s-1.mol-1 and 3.1 X 10(6) dm3.s-1.mol-1 respectively. The activities of the proteins are independent of the pH (7.4-9.4) and are about three orders of magnitude less than that of the native (Cu,Zn)-protein. The rate constants for the initial interaction of O2-. with the Co-proteins were determined to be (1.5-1.6) X 10(9) dm3.s-1.mol-1; however, in the presence of phosphate, partial inhibition is apparent [k approximately (1.9-2.3) X 10(8) dm3.s-1.mol-1]. To account for the experimental observations, two reaction schemes are presented, involving initially either complex-formation or redox reactions between O2-. and Co(II). This is the first demonstration that substitution of a metal into the vacant copper site of (Cu,Zn)-protein results in proteins that retain superoxide dismutase activity.

Hydrogen-Ion Concentration↗
PubMed1982

Circulating immune complexes in rheumatoid arthritis. Selective removal by cryogelation with membrane filtration.

We have developed a system of extracorporeal circulation that removes proteins of the molecular weight of the circulating immune complexes of rheumatoid arthritis by cryogelation with hollow-fiber membrane filtration. A 52-year-old woman with a 36-year history of severe, unremitting, high-titer, seropositive rheumatoid arthritis who had failed to respond to anti-inflammatory, antirheumatic, and cytotoxic drugs was chosen for a trial of this system. A rapid and sustained decrease in circulating immune complexes as measured by C1q binding occurred, accompanied by a much slower improvement in clinical factors of disease activity. Rheumatoid factor changed very little and loss of other serum proteins by the procedure was relatively modest. This new procedure was successful in removing circulating immune complexes in a patient with rheumatoid arthritis, and in inducing a remission in one who has not had such in 36 years, while sparing volume and other plasma proteins.

Antigen-Antibody Complex↗
PubMed1981

Purification and properties of bovine caeruloplasmin.

A novel method is reported for isolation of bovine caeruloplasmin from plasma; it involves a rapid and mild procedure, namely two column chromatographies with stepwise elution and one (NH4)2SO4 precipitation, and results in a proteolytically undegraded homogeneous protein. The general structure of the protein, as evaluated by molecular-weight determination and amino acid composition, is very similar to that established for human and rat caeruloplasmin. Copper determination and e.p.r. spectral analysis on the native and NO-treated protein gave a metal-to-protein stoichiometry of six atoms of copper per molecule. Three copper atoms were detectable by e.p.r., with Type 2/Type 1 ratio = 1 : 3 in most samples. The protein is very sensitive to storage and/or handling. A component was isolated from aged samples, which was found to contain approximately four copper atoms per 125000 daltons, two of which were detectable by e.p.r. with the characters of Type 2 copper. However, the same component was found to be present, although to a lesser extent, in the fresh preparation and does not seem to be related to proteolytic degradation. This component has no oxidase activity. On the basis of these results it is suggested that caeruloplasmin molecules are intrinsically heterogeneous with respect to both copper content and copper type, and this can explain the intriguing stoichiometry regarding the different types of copper centres.

Amino Acids↗
PubMed1981

Preparation of selectively metal-free and metal-substituted derivatives by reaction of Cu--Zn superoxide dismutase with diethyldithiocarbamate.

Incubation of Cu--Zn superoxide dismutase with diethyldithiocarbamate at increasing ligand/protein ratios and subsequent high-speed centrifugation led to proportional removal of copper from the protein, at variance with previous results [Misra (1979) J. Biol. Chem. 254, 11623--11628]. No zinc was lost, even at very high excesses of chelating agent. In this way a copper-free protein could be readily prepared, with avoidance of the critical pH condition and the dialysis step required in a previous method employing cyanide. The holoprotein was fully reconstituted from the copper-free protein by stoicheiometric re-addition of copper. From the mixture of metal-depleted forms originated by treatment with slight diethyldithiocarbamate excess, the protein containing copper only on one subunit, [Cu1--Zn2], could be isolated by preparative column electrophoresis. This species reproducibly showed 25% more specific activity (catalytic constant per copper) than that of the native or reconstituted [Cu2--Zn2] protein. This may result from long-range conformational effects between the active sites. By adding Co2+ ions to the vacant copper site of [Cu1--Zn2] a hybrid molecule containing Cu(II) on one subunit and Co(II) in the homologous site of the other subunit was prepared. Its activity, referred to copper, was identical with that of the native protein.

Binding Sites↗
PubMed1981

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins.

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.

Alcohol Oxidoreductases↗
PubMed1981

Re-examination of the reaction of diethyldithiocarbamate with the copper of superoxide dismutase.

The reaction of the copper of (Cu,Zn)-superoxide dismutase with diethyldithiocarbamate was studied at pH = 7.4 and the results obtained led to a reaction scheme basically different from the conclusion of a previous study (Misra, H. P. (1979) J. Biol. Chem. 254, 11623-11628). The analysis of optical and ESR spectra at 9 and 35 GHz, at different ligand/protein ratios and reaction times, showed that a ternary diethyldithiocarbamate. Cu(II).protein complex never formed in spectroscopically detectable amounts. The system is described in any condition as the mixture, in variable proportions, of only two components, that is the diethyldithiocarbamate-free (Cu(II) chelate and the copper-depleted protein. The formation of a catalytically active copper-diethyldithiocarbamate intermediate with distinct optical and ESR spectra was also ruled out by kinetic studies, which demonstrated that enzyme inactivation strictly parallels the binding of diethyldithiocarbamate as monitored by optical absorption and ESR. Separation of the copper complex from the protein was obtained for the first time, and the procedure was suitable for rapid preparation of reconstitutable copper-free superoxide dismutase.

Animals↗
PubMed1981

Dependence on freezing of the geometry and redox potential of type 1 and type 2 copper sites of Japanese-lacquer-tree (Rhus vernicifera) laccase.

The room-temperature e.p.r. spectrum of the Japanese-lacquer-tree (Rhus vernicifera) laccase shows A parallel (the hyperfine splitting constant) and g parallel values of both the Type 1 and Type 2 Cu appreciably different from those measured at liquid-N2 temperature. The geometry of the sites, as inferred from the room-temperature e.p.r. parameters, is more consistent with their redox properties. A rough correlation is found between A parallel and g parallel values and redox potential of the blue copper in several enzymes.

Chemical Phenomena↗
PubMed1981

Primary ultrastructural diagnosis of cryoglobulinemic glomerulonephritis.

A 58-year-old woman suffering from epistasis was found to be hypertensive. Hypocomplementemia associated with normal serum creatinine level, erythrocytosis, microscopic hematuria, proteinuria, and normal serum immunoglobulin levels and immunoelectrophoresis results led to a renal biopsy. Light microscopy disclosed mesangial hypercellularity and accumulation of eosinophilic material in capillary loops and in the mesangium. Immunomicroscopy disclosed coarsely granular to globular deposition of IgG, IgM, and C3 in similar distribution. Electron microscopy demonstrated electron-dense mesangial and subendothelial deposits consisting of interlacing fibrillar bundles associated with infiltration of acute inflammatory cells characteristic of cryoglobulinemic glomerulonephritis. After the biopsy, the presence of circulating IgG-IgM cryoglobulins was documented. Substructure of the in vitro cryoprecipitate and the immune complexes in the kidney was very similar. These observations illustrate the importance of continued routine ultrastructural assessment in evaluation of renal biopsy material.

Cryoglobulins↗