Quantitative isolation of liver mitochondria by zonal centrifugation

A method was developed using zonal centrifugation to recover liver mitochondria quantiatively and free of other cellular components from a sample of whole homogenate. The fractions containing mitochondria were identified by the distribution of cytochrome oxidase and these fractions contained over 90% of the total cytochrome oxidase recovered. The mitochondrial fractions were found to be only slightly contaminated by 5′-nucleotidase (plasma membranes), acid phosphatase (lysosomes), glucose-6-phosphatase (microsomes), and catalase (peroxisomes). There was no detectable contamination by nuclear DNA (nuclei). This method was used to quantitate total liver mitochondrial protein. The development of this procedure provides a means for following total changes in mitochondrial components during mitochondrial biogenesis.     

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.     

Theory and practice of the analytical centrifugation of an active substrate-enzyme complex

The active enzyme centrifugal-ion (AEC) method presented here permits the hydrodynamic study of active enzyme–substrate(s) complexes in solutions containing micro-gram amounts of the studied enzyme, even if the enzyme preparation is very impure. The AEC method can he used only when the specific enzymatie reaction can be measured directly in a spectrophotometer. The general equations relevant to the method and their solutions are presented in detail. Their use requires some numerical calculations. A practical summary of the AEC method is given, and the precision of the measured values of the sedimentation and diffusion coefficients is discussed.     

Loss of translational entropy in molecular associations

Molecular associations in solution are opposed by the loss of entropy (DeltaS) that results from the restriction of motion of each component in the complex. Theoretical estimates of DeltaS are essential for rationalizing binding affinities, as well as for calculating entropic contribution to enzyme catalysis. Recently a statistical-mechanical framework has been proposed for estimating efficiently the translational entropy loss (DeltaS(trsl)), while taking explicitly into account the complex intermolecular interactions between the solute and the solvent. This framework relates the translational entropy of a solute in solution to its "free volume," defined as the volume accessible to the center of mass of the solute in the presence of the solvent and calculated by using an extension of the cell model (CM) for condensed phases. The translational entropy of pure water, estimated with the CM algorithm, shows good agreement with the experimental information. The free volume of various solutes in water, calculated within the CM by using molecular dynamics simulations with explicit solvent, displays a strong correlation with the solutes' polar and total surface areas. This correlation is used to propose a parameterization that can be used to calculate routinely the translational entropy of a solute in water. We also applied the CM formalism to calculate the free volume and translational entropy loss (DeltaS(trsl)) on binding of benzene to a cavity in a mutant T4-lysozyme. Our results agree with previously published estimates of the binding of benzene to this mutant T4-lysozyme. These and other considerations suggest that the cell model is a simple yet efficient theoretical framework to evaluate the translational entropy loss on molecular association in solution.     

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.     

Predicting disease associations via biological network analysis

Background

Understanding the relationship between diseases based on the underlying biological mechanisms is one of the greatest challenges in modern biology and medicine. Exploring disease-disease associations by using system-level biological data is expected to improve our current knowledge of disease relationships, which may lead to further improvements in disease diagnosis, prognosis and treatment.

Results

We took advantage of diverse biological data including disease-gene associations and a large-scale molecular network to gain novel insights into disease relationships. We analysed and compared four publicly available disease-gene association datasets, then applied three disease similarity measures, namely annotation-based measure, function-based measure and topology-based measure, to estimate the similarity scores between diseases. We systematically evaluated disease associations obtained by these measures against a statistical measure of comorbidity which was derived from a large number of medical patient records. Our results show that the correlation between our similarity measures and comorbidity scores is substantially higher than expected at random, confirming that our similarity measures are able to recover comorbidity associations. We also demonstrated that our predicted disease associations correlated with disease associations generated from genome-wide association studies significantly higher than expected at random. Furthermore, we evaluated our predicted disease associations via mining the literature on PubMed, and presented case studies to demonstrate how these novel disease associations can be used to enhance our current knowledge of disease relationships.

Conclusions

We present three similarity measures for predicting disease associations. The strong correlation between our predictions and known disease associations demonstrates the ability of our measures to provide novel insights into disease relationships.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-304) contains supplementary material, which is available to authorized users.     

The characterization of rat kidney plasma membranes prepared by zonal centrifugation

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.     

Quantitative determination and reversible modification of sulfhydryl groups in low and high molecular weight compounds using a biradical spin marker]

A new method for quantitation of sulfhydryl groups of low and high molecular weight compounds is proposed. The method is based on the use of a biradical spin label carrying a disulfide bond, RS-SR, where R is the imidazoline radical. It was found that this biradical is involved in the reaction of thiol-disulfide exchange with thiols; the EPR spectra of the original biradical and monoradical products differ essentially. This circumstance made it possible to determine the bimolecular rate constant for the biradical interaction with cysteamine, cysteine, glutathione and human serum albumin. The method was used for measuring glutathione and cysteine levels in murine and rat blood and for assaying the insect acetylcholine esterase activity and reversible inhibition of NADPH-cytochrome P-450 reductase. The method is marked for a high sensitivity (10(-6)-10(-7) M) towards sulfhydryl groups and allows the determination of thiol groups in coloured and nontransparent solutions.     

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.     

Determination of small differences in buoyant density of macromolecules by analytical density gradient equilibrium centrifugation.

A sensitive method is proposed for the determination of small differences between the buoyant densities of different species of monodisperse macromolecules by analytical density gradient equilibrium centrifugation. The procedure involves the measurement at sedimentation equilibrium of the bandwidths of the concentration distribution of the separate macromolecules and of a mixture of the different species. The difference in buoyant densities can then be estimated from the difference between the bandwidths.     

Characterization of pinocytic vesicles from CHO cells: resolution of pinosomes from lysosomes by analytical centrifugation

Pinocytic vesicles (pinosomes) and lysosomes from suspension cultured, Chinese hamster ovary (CHO-S) cells have been resolved as two non-overlapping organelle populations by analytical centrifugation in Percoll gradients. Pinosomes were labeled with either horseradish peroxidase (HRP), a fluid phase content marker, or by radioiodination by pinocytosed lactoperoxidase (LPO). CHO-S cell lysosomes followed by three different marker enzymes and electron microscopy behaved as a single, dense organelle population. Pinosomes were partially resolved from plasma membrane, a less dense organelle population.     

Identification and characterization of a new reversible MAGL inhibitor

Monoacylglycerol lipase is a serine hydrolase that play a major role in the degradation of 2-arachidonoylglycerol, an endocannabinoid neurotransmitter implicated in several physiological processes. Recent studies have shown the possible role of MAGL inhibitors as anti-inflammatory, anti-nociceptive and anti-cancer agents. The use of irreversible MAGL inhibitors determined an unwanted chronic MAGL inactivation, which acquires a functional antagonism function of the endocannabinoid system. However, the application of reversible MAGL inhibitors has not yet been explored, mainly due to the scarcity of known compounds possessing efficient reversible inhibitory activities. In this study we reported the first virtual screening analysis for the identification of reversible MAGL inhibitors. Among the screened compounds, the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) is a promising reversible MAGL inhibitor lead (K_i = 8.6 μM), which may be used for the future development of a new class of MAGL inhibitors. Furthermore, the results demonstrate the validity of the methodologies that we followed, encouraging additional screenings of other commercial databases.     

Modulating molecular chaperone Hsp90 functions through reversible acetylation

The molecular chaperone protein Hsp90 is a key regulator of approximately 100 'client' proteins crucial for numerous cell signaling processes. Consequently, understanding the molecular underpinnings that regulate Hsp90 activity is an important biological endeavor. Exciting new results now suggest that, at least for nuclear receptor activity, Hsp90 function is directly regulated by histone deacetylase 6 (HDAC6). These observations have consequences for various biological processes and potentially important implications for the development of cancer therapeutics.