Identification of mitochondrial protein complexes inArabidopsis using two-dimensional blue-native polyacrylamide gel electrophoresis

Mitochondria fulfill a wide range of functions in the plant cell, including producing ATP, providing carbon skeletons for biosynthesis, and biosynthesizing vitamins and cofactors. Recently, mitochondria have been implicated in the pathway of programmed cell death in plant cells. In addition, mutations in the mitochondrial genome have been shown to be causally related to cytoplasmic male sterility—the failure to produce functional pollen in a range of crop plants. Proteomics has been used to attempt to catalogue mitochondrial proteins and extend our understanding of this essential organelle. Conventional proteomics based on isoelectric focusing and SDS-PAGE is unsuitable for hydrophobic proteins and therefore does not allow the identification of many components of the respiratory complexes. To identify such proteins, we have used blue-native PAGE to fractionate protein complexes in their native state, followed by SDS-PAGE to separate component subunits of each complex. A total of 40 protein spots were reproducibly resolved, and 29 were identified by means of mass spectrometry, thus giving a map of the most abundant complexes in plant mitochondria. Chaperones; transporters; novel proteins; and proteins involved in the respiratory chain, the citric acid cycle, amino acid and carbon metabolist, and stress response were identified. This study gives new insight on the role and functioning of well-characterised and recently identified mitochondrial proteins by localising them to specific complexes. It also identifies novel proteins in plant mitochondria.     

Improved retention of heme with increased resolution of microsomal proteins in polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis, using lithium dodecyl sulfate instead of the sodium salt, was used to analyze rat hepatic microsomal hemoproteins. Good resolution of hepatic microsomal proteins was obtained with retention of approximately half of the total microsomal heme on the proteins with molecular weights of 45,000 to 50,000. Both the protein resolution and heme retention are better than with electrophoresis procedures previously described. Treatment of rats with chemicals that either increase or decrease microsomal cytochrome P-450 produced proportional changes in the heme associated with the proteins of 45,000 to 50,000 molecular weights on the gel.     

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.     

检测SDS-聚丙烯酰胺凝胶电泳中家蝇蛋白的新方法——海波银染法

介绍一种检测SDS聚丙烯酰胺凝胶电泳中家蝇幼虫蛋白的新方法-海波银染法。该方法对传统银染方法中的试剂与步骤加以改进,省略了乙醇固定与洗涤步骤,只需20 min即可完成全部染色过程,且仅在国产分析纯试剂及普通操作条件下,灵敏度可达毫微克级水平。     

Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis

Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds.     

The use of gel electrophoresis to study the reactions of activated amino acids with oligonucleotides

We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonucleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5-phosphates and ethylenediamine. We find that arginine and amino acids that can interact with oligonucleotides through stacking interactions react most efficiently. D-and L-amino acids give indistinguishable families of products.Correspondence to: L.E. Orgel 1444     

Improved resolution with one-dimensional polyacrylamide gel electrophoresis: myofibrillar proteins from typed single fibers of human muscle

Standard procedures for one-dimensional discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining were modified to give more effective separation and an improved resolution of human skeletal muscle proteins. In this system, an electrophoresis buffer composed of 100 mM L-isoleucine, 25 mM Tris base, and 0.1% SDS was used. The separating gel consisted of 16% acrylamide with N,N'-methylenebisacrylamide as a crosslinker (1:23), 0.4% SDS, 1.5 M Tris-HCl, pH 8.8. By the present procedure, the slow and the fast forms of myosin light chains (LCs, LCf) and other contractile proteins from human muscle could be better separated. The silver stain is based on a combination of methods previously described. The modified method requires a small fragment of a single fiber to observe as few as 10 ng of myofibrillar muscle proteins. The described simplifications made it possible to assay and compare up to 40 single fibers in the same electrophoretic run. Improved separation of other proteins migrating at basic pH could be achieved by a similar approach.     

Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride

Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen with the unaided eye so that the separation during electrophoresis could be followed. Dabsylation did not affect the mobility of the proteins. The location of the orange band gave a good indication of the position of the protein in the gel so that molecular weight estimations could be made during and immediately following electrophoresis.     

Inhibition of invitro sickling by liposome-mediated transport of amino acids into intact human red blood cells

To investigate the role of phenylalanine and tryptophane as potential antisickling agents in intact human SS-red blood cells a liposomal transport system was employed to transfer phenyl-alanine or tryptophane into intact SS-red blood cells. Aromatic amino acids and short peptides containing phenylalanine have been demonstrated to increase the minimum gelling concentration and solubility of deoxy-hemoglobin S in aqueous solution. However, these compounds do not cross the red blood cell membrane under usual incubation conditions. Incorporation of phenylalanine or tryptophane into intact SS-red blood cells $\text{via}$ liposomal transport system markedly inhibited the $\text{in}\text{vitro}$ sickling of deoxy-hemoglobin S. These findings raise the possibility that a nontoxic liposomal transport system which facilitates incorporation of antisickling agents into intact SS-RBC may have significant therapeutic implications in the treatment of sickle cell disease.     

Increasing the resolution of polyacrylamide gel electrophoresis by varying the degree of gel crosslinking

Resolution of polyacrylamide gel electrophoresis may be substantially improved by taking advantage of the gel sieving effects of varying concentrations of bisacrylamide crosslinker. A dilution procedure is described which permits simultaneous variation of both total acrylamide concentration and percent crosslinking within a single linear regression analysis.This work was supported by NSF Grant 10584 and NIH Grant 23504.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis as a method for studying the stability of subtilisin

A simple method has been developed to study the stability of subtilisin. Protein incubated at various temperatures in the presence of proteinase inhibitor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and showed a transition from the intact state to the unfolded state between 55 degrees C and 65 degrees C. Additionally, autolysis was also observed above 65 degrees C. In the absence of inhibitor, similar results were obtained below 55 degrees C; however, above 65 degrees C no protein of any size was observed due to extensive autolysis. These results demonstrate that SDS-PAGE can trap subtilisin in the state in which the protein existed prior to the analysis. It can be used to identify the different forms, including autolysis products, of the protein generated by heat denaturation. This method was used to study SDS-induced unfolding of aprA-subtilisin. When the protein was incubated with 0.25% SDS at different NaCl concentrations, a gradual increase in unfolding was observed with increasing NaCl concentration. This change paralleled a decrease in the critical micelle concentration of SDS, indicating that the rate of unfolding of aprA-subtilisin increases with increasing SDS micelle concentration. No detectable unfolding was observed below the critical micelle concentration.     

New procedure of high-voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids.

Addition of primary organic amines, such as n-butylamine, to the mobile phase altered the capacity factors and selectivity of benzo[a]pyrene metabolites obtained with reverse-phase high pressure liquid chromatography on an ODS column. Separation of benzo[a]pyrene phenols in particular was improved with 8 of the 10 available metabolites resolved, including those known to be biologically produced. The method offers sufficiently improved resolution or convenience that it should prove useful in comparative studies of metabolism of benzo[a]-pyrene and other polynuclear aromatic hydrocarbons. Applying the method to analysis of benzo[a]pyrene metabolites produced in vitro by hepatic microsomes from the marine fish Stenotomus versicolor indicated the principal phenolic derivatives produced by this fish were 1-hydroxy-, 3-hydroxy-, 7-hydroxy-, and 9-hydroxybenzo[a]pyrene.     

Data standards for proteomics: mitochondrial two-dimensional polyacrylamide gel electrophoresis data as a model system

Proteomics has emerged as a major discipline that led to a re-examination of the need for consensus and a nationally sanctioned set of proteomics technology standards. Such standards for databases and data reporting may be applied to two-dimensional polyacrylamide gel electrophoresis (2D PAGE) technology as a pilot project for assessing global and national needs in proteomics, and the role of the National Institute of Standards and Technology (NIST) and other similar standards and measurement organizations. The experience of harmonizing the heterogeneous data included in the Protein Data Bank (PDB) provides a paradigm for technology in an area where significant heterogeneity in technical detail and data storage has evolved. Here we propose an approach toward standardizing mitochondrial 2D PAGE data in support of a globally relevant proteomics consensus.     

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide gel electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AU-PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.     

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis

Blue native gel electrophoresis (BN–PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN–PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN–PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed.