Indépendance des formes spectrales de chlorophylle a et des holochromes chlorophylliens

Independence of special forms of chlorophyll a and chlorophyll holochromesZea mays L. seedlings were cultivated for 10 days with submission to 4 s illumination periods interspersed with dark periods varying in length from 30 min to 6 h depending on the lot analyzed. The results show that, for the case in which the dark periods were shorter than 1 h, the relative proportions of different spectroscopic chlorophyll forms (maxima at 662, 670, 677.5, and 684 nm) were constant. For longer durations of darkness between illuminations, the relative proportion of the form Ca₆₇₀ increases, while that of Ca₆₈₄ diminishes with the length of darkness; to a lesser extent, the relative proportion of Ca₆₆₂ increases and a form Ca₆₉₂ disappears. A scheme is proposed to explain the evolution of the relative proportions of the different spectral forms.The different chlorophyll holochromes present in the chloroplasts were also analysed. If the dark period was longer than 1 h, chlorophyll was associated with peptide chains of molecular weights 21 000 and 29 000. If the dark period was shorter than 1 h chlorophyll was associated with four peptide chains of molecular weights 21 000, 25 000, 29 000 and 70 000.The results taken together demonstrate that a given spectral chlorophyll a form cannot be associated with a definite chlorophyll holochrome.     

Selective extraction of CP 26 and CP 29 proteins without affecting the binding of the extrinsic proteins (33, 23 and 17 kDa) and the DCMU sensitivity of a Photosystem II core complex

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl--D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC light-harvesting complex - MES 2-(N-morpholino)ethanesulfonic acid - OGP n-octyl--d-glucoside - OTG n-octyl--d-thioglucoside - PAGE polyacrylamide gel electrophoresis - PS II Photosystem II - SDS sodium dodecyl sulfate     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle

In microsomal vesicles isolated from several smooth muscles many polypeptides were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. In pig stomach and in rabbit and dog aorta components of M_r 22 000 and 11 000 were identified as forms of phospholamban. These polypeptides were, however, not observed in pig aorta. These phospholamban-like polypeptides presented the same electrophoretic mobility in sodium dodecyl sulphate gels as cardiac phospholamban, and the 22 000 M_r form showed a similar reaction to heat treatment in sodium dodecyl sulphate. Antibodies against purified canine cardiac phospholamban cross-reacted with the 22 000 and 11 000 M_r phosphorylatable polypeptides from smooth muscle membranes. Subcellular fractionation of porcine stomach smooth muslce indicated that phospholamban was present in the membranes of the endoplasmic reticulum and not in the plasma membranes. Phospholamban was also phosphorylated by an endogenous calcium—calmodulin-dependent protein kinase and by an endogenous cyclic AMP-dependent kinase. It is concluded that the endoplasmic reticulum of many, but possibly not all, smooth muscles contains phospholamban. However, the physiological role of phospholamban in smooth muscle remains to be established.     

Isolation and characterization of a major chlorophyll ac2 light-harvesting protein from a Chroomonas species (Cryptophyceae)

Three chlorophyll-protein complexes of a Chroomonas species (Cryptophyceae) have been separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The two bands at 100 and 42 kDa are Complex I (CP I) and Complex IV (CP IV), the ubiquitous chlorophyll a-proteins associated with Photosystems I and II, respectively. The third 55 kDa band, which had two peptide subunits (24 and 20 kDa), contained both chlorophyll a and chlorophyll c₂ in a molar ratio of 1.4 chlorophyll a to 1 chlorophyll c₂ ($\text{chlorophyll}\text{a}\text{chlorophyll}\text{c}{}_2$ ratio in whole cells = 4). A chlorophyll $\text{a}\text{c}{}_2$ fraction with similar spectral and electrophoretic properties was isolated by digitonin-sucrose density gradient centrifugation. This fraction had no photochemical activity and contained only a single carotenoid species with absorbance maxima in methanol at 424, 448 and 476 nm. Efficient energy transfer from chlorophyll c₂ to chlorophyll a occurred in the complex.     

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

The structure and heterogeneity of LHC II were studied by in vitro reconstitution of apoproteins with pigments (Plumley and Schmidt 1987, Proc Natl Acad Sci 84: 146–150). Reconstituted CP 2 complexes purified by LDS-PAGE were subsequently characterized and shown to have spectroscopic properties and pigment-protein compositions and stoichiometries similar to those of authentic complexes. Heterologous reconstitutions utilizing pigments and light-harvesting proteins from spinach, pea and Chlamydomonas reinhardtii reveal no evidence of specialized binding sites for the unique C. reinhardtii xanthophyll loroxanthin: lutein and loroxanthin are interchangeable for in vitro reconstitution. Proteins modified by the presence of a transit peptide, phosphorylation, or proteolytic removal of the NH₂-terminus could be reconstituted. Evidence suggests that post-translational modification are not responsible for the presence of six electrophoretic variants of C. reinhardtii CP 2. Reconstitution is blocked by iodoacetamide pre-treatment of the apoproteins suggesting a role for cysteine in pigment ligation and/or proper folding of the pigment-protein complex. Finally, no effect of divalent cations on pigment reassembly could be detected.Abbreviations cab chlorophyll a/b-binding protein genes - Chl chlorophyll - CP2 light-harvesting chlorophyll A+b-protein complex fractionated by mildly denaturing LDS-PAGE from Photosystem II in thylakoids - CP 43 and CP 47 chlorophyll a-antenna complexes fractionated from Photosystem II in thylakoids by mildly denaturing LDS-PAGE at 4°C - IgG gamma immunoglobulin - LDS lithium dodecyl sulfate - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C - LHC I and LHC II thylakoid light-harvesting chlorophyll a+b-protein holocomplexes associated with Photosystems I and II, respectively - PS II Photosystem II - TX100 Triton X-100 - TX100-derived LHC light-harvesting complexes enriched in LHC II following fractionation of thylakoids by TX100     

Protein sequences and redox titrations indicate that the electron acceptors in reaction centers from heliobacteria are similar to Photosystem I

Photosynthetic reaction centers isolated from Heliobacillus mobilis exhibit a single major protein on SDS-PAGE of 47 000 M_r. Attempts to sequence the reaction center polypeptide indicated that the N-terminus is blocked. After enzymatic and chemical cleavage, four peptide fragments were sequenced from the Heliobacillus mobilis apoprotein. Only one of these sequences showed significant specific similarity to any of the protein and deduced protein sequences in the GenBank data base. This fragment is identical with 56% of the residues, including both cysteines, found in the highly conserved region that is proposed to bind iron-sulfur center F_X in the Photosystem I reaction center peptide that is the psaB gene product. The similarity to the psaA gene product in this region is 48%.Redox titrations of laser-flash-induced photobleaching with millisecond decay kinetics on isolated reaction centers from Heliobacterium gestii indicate a midpoint potential of –414 mV with n=2 titration behavior. In membranes, the behavior is intermediate between n=1 and n=2, and the apparent midpoint potential is –444 mV. This is compared to the behavior in Photosystem I, where the intermediate electron acceptor A₁, thought to be a phylloquinone molecule, has been proposed to undergo a double reduction at low redox potentials in the presence of viologen redox mediators.These results strongly suggest that the acceptor side electron transfer system in reaction centers from heliobacteria is indeed analogous to that found in Photosystem I. The sequence similarities indicate that the divergence of the heliobacteria from the Photosystem I line occurred before the gene duplication and subsequent divergence that lead to the heterodimeric protein core of the Photosystem I reaction center.Abbreviations BChl bacteriochlorophyll - %C percent bisacrylamide as a percentage of total acrylamide - DTT dithiothreitol - EPR electron paramagnetic resonance - Fe-S iron-sulfur center - H. Heliobacterium - Hb. Heliobacillus - k one thousand - M_r molecular retention - PS I Photosystem I - PS II Photosystem II - RCs reaction centers - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide electrophoresis - %T percent total acrylamide - Tris tris(hydroxymethyl)aminomethane     

Purification and characterization of laccase from Monocillium indicum Saxena

Summary An ascomycete Monocillium indicum Saxena producing extracellular laccase was isolated. The culture filtrate on native polyacrylamide gel electrophoresis (PAGE) revealed four bands of activity, one of which was a major one. The major laccase band, a glycoprotein, was purified and characterized. Gel filtration chromatography showed that the relative molecular weight (M_r) of laccase was 100 000. On sodium dodecyl sulphate (SDS)-PAGE the major laccase band further resolved into three proteins of M_r 72 000, 56 000 and 24 000. The enzyme had a pH optimum of 3.0 and was active on a number of o-phenols and aromatic acids. The 72 000 M_r protein was found to share common immunological properties with laccases of Coriolus versicolor, Agaricus bisporus and lignin peroxidase of Phanerochaete chrysosporium. Correspondence to: K. Koteswara Rao     

Chloroplast thylakoid membrane proteins having buried amine buffering groups

Some chloroplast thylakoid membrane proteins have anomalously low pK_a (near 7.8) amine groups, indicating that the buffering groups may be buried in hydrophobic regions and/or close to other positive charges. Other work has shown that the low pK_a amine group array is not in ready equilibrium with either the inner or outer bulk aqueous phases (Laszlo, J.A., Baker, G.M. and Dilley, R.A. (1984) J. Bioenerg. Biomembranes, 16, 37–51). Acetic anhydride reacts with the neutral amine and has been used as a probe for labeling the low pK_a amines. The buried array of buffering groups can be labeled with [³H]acetic anhydride in the dark only after the membranes were made leaky to protons with uncoupler addition. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis was used to separate the polypeptides and identify those that show the uncoupler-dependent labeling increase. Included in that group are polypeptides known to be associated with Photosystem II having M_r 17000, 22000 and 31000, some of the light-harvesting pigment proteins with M_r 24000–28000, the CF₀ component with M_r 8000 and some polypeptides associated with Photosystem I. A protein with M_r 15000 showed very large changes in labeling, but the identity of this polypeptide is unknown. The arrays of buried amine buffering groups are diversely distributed among membrane proteins and it is not clear what role, if any, they play in membrane function.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, $\text{O-(α-2-}\text{sulphamino}\text{-2-}\text{deoxy-}\text{D}\text{-glucopyranosyl}\text{)-(1→3)-}\text{L}\text{-[6,}{}^3\text{H]}\text{idonic}$ acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent M_r of the haloenzyme as determined by gel filtration was 190 000 ± 20 000. Two other larger M_r protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoreced as a single major band with an apparent M_r corresponding to 55 000 ± 6000.     

Tissue inhibitor of metalloproteinases. Identification of precursor forms synthetized by human fibroblasts in culture

Precursor forms of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) synthesized by human fibroblasts in culture have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of specific immunoprecipitates. Translation of mRNA extracted from fibroblasts in the cell-free rabbit reticulocyte lysate system yielded a single immunoprecipitable precursor of tissue inhibitor of metalloproteinases, M_r 22 000. Intact fibroblasts cultured in the presence of tunicamycin synthesized an M_r 20 000 form of tissue inhibitor of metalloproteinases, detectable intracellularly and extracellularly. This is in contrast to the predominantly intracellular M_r 24 000.form synthetized during monensin treatment of cells and the normal secreted form of tissue inhibitor of metalloproteinases, M_r 29 000. Isoelectric focusing of the various immunoprecipitable precursor forms showed a progressive increase in positive charge and microheterogeneity of the protein during cellular processing. The data suggest that the inhibitor protein core, of basic pI, is glycosylated initially by the addition of mostly neutral sugars and subsequently by acidic sugars, prior to secretion.     

Isolation of human platelet glycoproteins

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of M_r ≈ 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of M_r ≈ 165 000.Treatment of whole platelets by periodate oxidation and sodium[³H]borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of M_r ≈ 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with M_r ≈ 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others.Phosphorylation of intact platelets with ³²P_i also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the     

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem I CP complexes were observed before the photosystem II and light-harvesting CP complexes during the initial stages of barley chloroplast development. Photosystem I activity was observed before the photosystem I CP complex was detected whereas photosystem II activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem I remained constant whereas the amount of chlorophyll associated with photosystem II and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - CP A/B the major light-harvesting complex associated with photosystem II - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenyl carbazide - MV methyl viologen - PAR photosynthetically active radiation - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TEMED N,N,N,N-tetramethylethylenediamine - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 9949 of the Journal Series of the North Carolina Agricultural Research Service, Raleight, NC 27695-7601.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures

Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 ± 5000, 105,000 ± 5000, and 94,000 ± 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M_r, as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K_m values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and α-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.     

Lysine N-hydroxylase and N-acetyltransferase of the aerobactin system of pColV plasmids in Escherichia coli

The time course of formation of lysine N-hydroxylase and N-transacetylase in Escherichia coli bearing cloned genes derived from pColV-K311 was followed and the influence of iron concentration on the enzyme induction was studied. Specific activities of both enzymes were determined for 8 strains prepared by Braun and coworkers [1,2] comprising the separated genes on the vectors pBR322 or pACYC184. The assignment of genes aerA and aerB to the first two enzymes of aerobactin biosynthesis [2] was confirmed. The active enzyme encoded by aerA (N-hydroxylase) has an M_r of 52 000 and that of aerB (N-transacetylase) an M_r of 70 000, as determined by gel filtration. The M_r of the N-transacetylase was 35 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).     

Multiple forms of glucocorticosteroid receptors in the neural retina of chick embryo,revealed by polyacrylamide gel electrophoresis

The glucocortiocoid receptors in the cytosol of neural retina of the 15-day chick embryo were analyzed by quantitative polyacrylamide gel electrophoresis. Maintenance of the triamcinolone acetonide (TA)-receptor complexes under conditions of electrophoretic analysis is dependent on temperatures not exceeding ?2 °C and is favored by low ionic strength, but is relatively insensitive to changes in pH between 5 and 10. Polyacrylamide gel electrophoresis in highly crosslinked Resolving Gels (15% crosslinking with N,N′-diallyltartardiamide) at low wattage and under temperature control at ?2 °C, allowed for detection and partial characterization of over 80% of the specific TA-binding activity of the tissue. One form of the glucocorticoid receptor, designated as complex II, was found to have a molecular weight (M_r) of 175,000. In addition, specifically bound TA was found in a multimillion M_r aggregate which was unable to enter gels of any concentration investigated and has been designated TA-complex I. The ratio of complex I/complex II increased with increasing gel concentration, indicating physical or chemical interaction between II and I. A polyacrylamide gel electrophoresis rerun of isolated TA-complex II gave rise to two smaller TA-binding species: Component B, of M_r 108,000 and component A, a relatively fast migrating molecule which could not be characterized under the conditions used. The ratio of $\text{B}\text{A}$ appeared constant and close to 2, suggesting that A and B may be significant structural elements of complex II. Polyacrylamide gel electrophoresis of isolated TA-complex I gave rise to component C of M_r 60,000, but not to components A or B. Components A and B associated to a large M_r complex, designated as I′, which was revealed to an extent directly proportional to gel concentration. Similarly, component C aggregated to I″, as evidenced at elevated gel concentrations. In conclusion, it has been possible to define by gel electrophoresis three of the molecular species (A, B, and C) that comprise the glucocorticoid receptor, and the possible relationships between them.     

A Comparison of Pigment-Protein Complexes among Normal, Chlorophyll-Deficient and Senescent Soybean Genotypes

Pigment-protein complexes were isolated from chloroplasts of normal green and several types of chlorophyll-deficient soybeans. The complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparisons were made between normal and chlorophyll-deficient genotypes of the relative amounts of chlorophyll associated with Photosystem I (PSI), Photosystem II (PSII), light-harvesting, and free pigment complexes.

Chlorophyll-deficient genotypes, compared to normal green genotypes, have fewer light-harvesting complexes and a higher ratio of PSII to PSI complexes. Chlorophyll associated with PSII in yellow genotypes is in relatively higher amounts in spite of the fact that these genotypes have much less grana stacking than normal green genotypes. Although PSII activity has been associated with appressed regions of grana in normal plants, our work shows that the association does not always hold true.