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PubMed1981

Haptoglobin. A novel mode of biosynthesis of a liver secretory glycoprotein.

The de novo biosynthesis of the heterotetrameric (alpha 2 beta 2) serum glycoprotein, haptoglobin, was studied using a rabbit reticulocyte cell-free translation system and mRNA preparations from the livers of turpentine-treated rats. Analysis of the translation mixtures with either antisera specific for the alpha-subunit (anti-alpha-IgG) or the beta-subunit (anti-beta-IgG) or the native haptoglobin tetramer (anti-alpha 2 beta 2-IgG) resulted in each instance in the recovery of a single protein exhibiting a Mr = approximately 38,000. Cleavage of the translation product with cyanogen bromide or trypsin resulted in the generation of small peptide fragments that were specifically immunoadsorbed with either anti-alpha-IgG or anti-beta-IgG. These results indicated that the primary translation product of haptoglobin mRNA is a single polypeptide that contains the elements of both the alpha-subunit and the beta-subunit of the protein. Presumably haptoglobin is synthesized in vitro as a single precursor protein that is proteolytically processed post-translationally to form the dissimilar alpha- and beta-subunits of the native protein.

Animals↗
PubMed1985

Acclimation Processes in the Light-Harvesting System of the Cyanobacterium Anacystis nidulans following a Light Shift from White to Red Light.

Cyanobacteria acclimate to changes in light by adjusting the amounts of different cellular compounds, for example the light-harvesting macromolecular complex. Described are the acclimatization responses in the light-harvesting system of the cyanobacterium Anacystis nidulans following a shift from high intensity, white light to low intensity, red light.The phycocyanin and chlorophyll content and the relative amount of the two linker peptides (33 and 30 kilodaltons) in the phycobilisome were studied. Both the phycocyanin and chlorophyll content per cell increased after the shift, although the phycocyanin increased relatively more. The increase in phycocyanin was biphasic in nature, a fast initial phase and a slower second phase, while the chlorophyll increase was completed in one phase. The phycocyanin and chlorophyll responses to red light were immediate and were completed within 30 and 80 hours for chlorophyll and phycocyanin, respectively. An immediate response was also seen for the two phycobilisome linker peptides. The amount of both of them increased after the shift, although the 33 kilodalton linker peptide increased faster than the 30 kilodalton linker peptide. The increase of the content of the two linker peptides stopped when the phycocyanin increase shifted from the first to the second phase. We believe that the first phase of phycocyanin increase was due mainly to an increase in the phycobilisome size while the second phase was caused only by an increase in the amount of phycobilisomes. The termination of chlorophyll accumulation, which indicates that no further reaction center chlorophyll antennae were formed, occurred parallel to the onset of the second phase of phycocyanin accumulation.

Journal Article↗
PubMed2004

Electron-transfer reactions and functionalization of cytochrome P450cam monooxygenase system in reverse micelles.

Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media.

Camphor 5-Monooxygenase↗
PubMed1990

Model anionic polysaccharide matrices exhibit lower charge selectivity than is normally associated with kidney ultrafiltration.

The influence of the anionic polysaccharide matrix (APM) of the glomerular basement membrane (GBM) on water transport and macromolecular transport charge selectivity has been studied in model systems which enable the analysis of the specific properties of the APM. Various APMs were studied including heparin and heparin-like polysaccharides, chondroitin sulfate and dextran sulfate. Upper estimates of the APM concentration in the GBM were obtained by relating measurements of the specific hydraulic conductivity of the heparin-like polysaccharides to measurements of single nephron glomerular filtration rate. This gave values of 40-45 mg ml-1 of polysaccharide, which was then used to analyse factors contributing to macromolecular charge selectivity. Transport analysis of the test dextran probes, used for previous in vivo clearance studies, in APMs demonstrated that there were no differential charge effects at APM concentrations in the range predicted above in the GBM. This was also found to be the case for the partitioning of anionic test probes at the APM-solution interface as measured directly using frontal gel chromatography and through thermodynamic analysis of sedimentation and diffusion data. These studies demonstrated that previous biophysical interpretations of glomerular charge selectivity have severely overestimated the partitioning effect due to the electrostatic effects of polyion-polyion interaction.

Anions↗
PubMed1995

Properties of water-insoluble mitomycin C-albumin conjugate as a sustained release drug delivery system in mice inoculated with Sarcoma 180.

In order to improve the deposition of mitomycin C (MMC) in the administered site, water-insoluble mitomycin C-albumin conjugate (MMC-G-BSA) was prepared. MMC was covalently attached to the glutarylated bovine serum albumin (G-BSA) in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) to give MMC-G-BSA. The MMC content of the conjugate (16.3% (w/w)) was higher than that of water-soluble mitomycin C-albumin conjugates. MMC was liberated from MMC-G-BSA suspended in a phosphate buffer (pH 7.4, 37 degrees C) with a half-life of 155.3 h. In the same buffer system containing alpha-chymotrypsin, MMC-G-BSA was dissolved perfectly within 24 h due to enzymatic degradation, and the liberation of MMC from the conjugate was significantly accelerated (t1/2 = 24.5 h). After intraperitoneal injection in mice, most of the MMC-G-BSA was retained in the abdominal cavity. Furthermore, the survival time of mice inoculated with Sarcoma 180 was significantly increased by the intraperitoneal injection of MMC-G-BSA. These findings suggest that MMC-G-BSA is a biodegradable macromolecular hybrid which acts as a sustained release delivery system of MMC.

Albumins↗
PubMedDate not supplied

A comprehensive view of polyamine and histamine metabolism to the light of new technologies.

Polyamines and histamine are biogenic amines with multiple biological roles. In spite of the evidence for the involvement of both polyamines and histamine metabolism impairment in several highly prevalent pathological conditions, multiple questions concerning the molecular processes behind these effects remain to be elucidated. More comprehensive and systemic studies integrating molecular biology, biophysical and bioinformatics tools could contribute to accelerate the advances in this research area. This review is designed to underscore the main questions to be answered in polyamine and histamine research and how these new systemic approaches could help to find these answers.

Animals↗
PubMed2000

Large-scale shape changes in proteins and macromolecular complexes.

Proteins and RNA undergo intricate motions as they carry out functions in biological systems. These motions frequently entail large-scale conformational changes that induce changes in the surface structure, or shape, of a molecule. This review describes the experimental characterization of large-scale shape changes in proteins and macromolecular complexes and the effects of such changes on macromolecular behavior. We describe several important results that have been obtained by using small-angle scattering, which is emerging as a powerful technique for determining macromolecular shapes and elucidating the quaternary structure of macromolecular assemblies.

Animals↗
PubMed1985

Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body.

The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1975

Tetanus toxin: direct evidence for retrograde intraaxonal transport.

The neurotoxin tetanospasmin causes tetanus when it reaches the central nervous system. In this autoradiographic study, 125-I-labeled tetanospasmin was injected into the leg muscles of rodents, and the nerves supplying these muscles were crushed. The labeled toxin accumulated within axons on the distal side of the crush. This study provides direct evidence for retrograde axonal transport of a macromolecular toxin that acts at synapses in the central nervous system.

Animals↗
PubMed1988

Swelling-activated drug delivery systems.

A previous paper dealt with the preparation of an in vitro programmable zero-order drug delivery system in which the area of the surface exposed to the dissolution medium and the macromolecular relaxation of polymer controlled the release of the drug. In the present study, the preparation of similar delivery systems is described, in which differing drugs and polymers were used to ascertain the mechanism governing the drug-release kinetics. The movement of the interfaces between solvent and system was measured during drug release in systems with varying composition. The results indicate that the synchronization of the movement of swelling and eroding fronts at the solvent-system interface determines the achievement of the linear-release kinetics of such swelling activated systems and that the swelling and dissolution characteristics of the polymer employed for core preparation govern front movement.

Chemical Phenomena↗
PubMed1995

An early look at macromolecular crowding.

Interest in the interaction between proteins and polysaccharides in semidilute systems developed in the late 1950's and early 1960's both in the field of matrix biology and in the construction of new separation techniques. The author gives an account of how his work in the 50's on the characterization of a connective tissue polysaccharide, hyaluronan, led him into studies on polysaccharide networks, sieve effects, molecular exclusion, a theory of gel filtration, solubility of proteins and chemical equilibria in polymer solutions, water and protein homeostatis in vivo, and ordered convectional flow in concentrated polymer systems.

Journal Article↗
PubMed2004

[Microdevice system].

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Electrophoresis, Capillary↗