Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis

The size of hyaluronan in solid tissue was measured using a combination of agarose gel electrophoresis and a radiometric assay. Radiolabeled hyaluronan binding proteins, used in the radiometric assay, were also used to detect hyaluronan after transfer to a nylon membrane following gel electrophoresis. Lane intensity on the autoradiograph was linearly related to the amount applied to the gel between 10 and 100ng. The intensity was independent of the hyaluronan molecular weight for standards with molecular weights equal to or greater than 790,000. The radiometric assay was used to measure hyaluronan irrespective of size, while gel electrophoresis was used to measure hyaluronan with molecular weights greater than 0.79x10(6) or 4x10(6). Deferoxamine was used to inhibit depolymerization during the digestion of tissue samples with protease. The molecular weight pattern was similar for skin, skeletal muscle, heart, lung, small intestine, and large intestine despite large differences in hyaluronan content. For all tissues, 58% of the hyaluronan had a molecular weight greater than 4million. All tissues showed an absence of hyaluronan with a molecular weight below 790,000. The procedure can be used to study changes in hyaluronan size in tissue during inflammation and other pathological states.     

Measurement of DNA length by gel electrophoresis

Plotting fragment length against reciprocal of mobility gives a straight line over a wider range than the conventional semilogarithmic plot. Curvature can be removed by a simple correction. A method is also given for determining molecular weights from mobilities by direct calculation.     

Comparative analysis of plastocyanin–cytochrome f complex formation in higher plants,green algae and cyanobacteria

Mechanisms of the complex formation between plastocyanin and cytochrome f in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species of cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions force plastocyanin molecule close to the heme of cytochrome f. In the subsequent rotation of plastocyanin molecule around the point of electrostatic contact in the vicinity of cytochrome f, copper (Cu) atom approaches cytochrome heme forming a stable configuration where cytochrome f molecule behaves as a rather rigid body without conformational changes. In Nostoc plastocyanin molecule approaches cytochrome f in a different orientation (head‐on) where the stabilization of the plastocyanin–cytochrome f complex is accompanied by the conformational changes of the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation of the approaching molecules was not detected, thus indicating that random motions rather than long‐range electrostatic interactions are responsible for the proper mutual orientation. We demonstrated that despite the structural similarity of the investigated electron transport proteins in different photosynthetic organisms, the complexity of molecular mechanisms of the complex formation increases in the following sequence: non‐heterocystous cyanobacteria – heterocystous cyanobacteria – green algae – flowering plants.     

Statistical exploration of variation in quantitative two-dimensional gel electrophoresis data

Two-dimensional gel electrophoresis is a major technique in global analysis at the protein level. This paper presents an examination of spot volume data from three gel sets with radioactively labeled yeast Saccharomyces cerevisiae proteins. A strong variance versus mean dependence in data was found to be stabilized by applying a shifted logarithmic transformation. However, transformed data showed a remaining substantial variance heterogeneity for different proteins. Furthermore, examination of studentized residuals revealed that transformed data were approximately normally distributed and that there were spatial correlations among the measurement errors in the gel.     

An investigation of the degradation of cytochrome P-450 hemoproteins using SDS gel electrophoresis.

Treatment with fluroxene or allyl-$\text{iso}$-propylacetamide of rats induced for elevated levels of cytochromes P-450 results in markedly decreased levels of hepatic microsomal cytochromes P-450 and heme as determined by spectral assay but in unchanged levels of cytochromes P-450 as determined by SDS gel electrophoresis. Since SDS gel electrophoresis does not detect changes in the heme of cytochromes P-450, it is concluded that fluroxene and AIA do not chemically degrade the apoproteins of cytochromes P-450.     

Control of the photosynthetic electron transport by PQ diffusion microdomains in thylakoids of higher plants

We investigate the role of plastoquinone (PQ) diffusion in the control of the photosynthetic electron transport. A control analysis reveals an unexpected flux control of the whole chain electron transport by photosystem (PS) II. The contribution of PSII to the flux control of whole chain electron transport was high in stacked thylakoids (control coefficient, CJ(PSII) =0.85), but decreased after destacking (CJ(PSII)=0.25). From an 'electron storage' experiment, we conclude that in stacked thylakoids only about 50 to 60% of photoreducable PQ is involved in the light-saturated linear electron transport. No redox equilibration throughout the membrane between fixed redox groups at PSII and cytochrome (cyt) bf complexes, and the diffusable carrier PQ is achieved. The data support the PQ diffusion microdomain concept by Lavergne et al. [J. Lavergne, J.-P. Bouchaud, P. Joliot, Biochim. Biophys. Acta 1101 (1992) 13-22], but we come to different conclusions about size, structure and size distribution of domains. From an analysis of cyt b6 reduction, as a function of PSII inhibition, we conclude that in stacked thylakoids about 70% of PSII is located in small domains, where only 1 to 2 PSII share a local pool of a few PQ molecules. Thirty percent of PSII is located in larger domains. No small domains were found in destacked thylakoids. We present a structural model assuming a hierarchy of specific, strong and weak interactions between PSII core, light harvesting complexes (LHC) II and cyt bf. Peripheral LHCII's may serve to connect PSII-LHCII supercomplexes to a flexible protein network, by which small closed lipid diffusion compartments are formed. Within each domain, PQ moves rapidly and shuttles electrons between PSII and cyt bf complexes in the close vicinity. At the same time, long range diffusion is slow. We conclude, that in high light, cyt bfcomplexes located in distant stromal lamellae (20 to 30%) are not involved in the linear electron transport.     

The electrophoresis of histones in polyacrylamide gel and their quantitative determination

1. A new method has been devised for the separation of the histone fractions of calf thymus by electrophoresis in polyacrylamide gel at pH2.4. 2. The fractions have been characterized by their relative mobilities with respect to a marker protein, bovine plasma albumin. 3. A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye-histone complexes formed in the gel.     

The heterogeneity of phloem exudate proteins from different plants: A comparative survey of ten plants using polyacrylamide gel electrophoresis

Summary Proteins in sieve tube exudate from Ricinus communis L., Acer pseudoplatanus L., Aesculus hippocastanum L., Cucumis melo L., and two cultivars each of Cucumis sativus L., Cucurbita pepo L. and Cucurbita maxima Duchesne were fractionated and compared using polyacrylamide gel electrophoresis. Striking differences in major exudate proteins were displayed among the genera and species examined. Even cultivars within a single species, although showing general similarities, differed in some prominent proteins. Estimated molecular weights of the major exudate proteins from each plant are presented. The effects of reducing and chaotropic agents on the aggregation and subunit composition of exudate proteins from Cucumis sativus have been investigated. The problems involved in relating structure, function and biochemistry of P-protein are discussed.     

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension.

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.     

Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography

We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like architecture, but their size varies tremendously from approximately 15 × 30 nm to approximately 15 × 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.     

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

Electron structure of plastoquinone and coupling of electron and proton transport in thylakoids of the higher plants

The energy dependence on hydrogen position for a system, consisting of plastoquinone (in different redox states) and histidine molecules was studied. The distance between the atoms forming the hydrogen bond, an oxygen of the quinone molecule and a nitrogen of histidine, was supposed to be fixed. It was shown that for neutral quinone the total energy is minimal when the hydrogen is bound to histidine; for reduced quinone, the probability of hydrogen binding to quinone and histidine is approximately equal (so that a hydrogen bond is formed) and on secondary reduction of plastoquinone, the hydrogen binds to it.     

Apparent anomalies in the resolution of cytochrome P-450 isoenzymes by gel electrophoresis

Cytochromes P-450b and P-450e are two phenobarbital-inducible rat hepatic microsomal isoenzymes that possess approx. 97% sequence homology and show apparent anomalous behaviour in SDS-PAGE and isoelectric-focusing under denaturing conditions. Although the two enzymes differ in Mr by less than 0.1% and possess no apparent net charge difference, then can be resolved in both electrophoretic systems. These apparent discrepancies are discussed in terms of the predicted secondary structure of the proteins and the potential for certain specific amino acid substitutions to cause anomalous behaviour in SDS-PAGE. The results of electrophoresis of these cytochrome P-450 isoenzymes indicate that much smaller differences in primary structure can be detected by these methodologies than would be predicted from theoretical considerations.