Sodium Dodecyl Sulfate-polyacrylamide Disc Gel Research Articles

Expression of a truncated guanylate cyclase (GC-C), a receptor for heat-stable enterotoxin of enterotoxigenic Escherichia coli, and its dimer formation in COS-7 cells

A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method. The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125 I-N-5-azidonitrobenzoyl-STh[5-19]. Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells. The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C. Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system.

A phospholipase C with a high specificity for platelet-activating factor in rabbit liver light mitochondria.

The light mitochondrial fraction from rabbit liver was found to catalyze the hydrolysis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the phospholipase C reaction to form 1-O-alkyl-2-acetyl-glycerol and phosphocholine. The highest specific phospholipase C activity occurred in the liver and kidney. A subcellular survey showed that the enzyme was of lysosomal origin. The enzyme was solubilized with 2% Triton X-100 from rabbit liver light mitochondria and purified ca. 600- to 700-fold with a 17% yield using procedures that included hydroxyapatite, Sepharose 4B and isoelectric focusing column chromatography followed by fast protein liquid chromatography. The enzyme consists of two forms having a pl of 4.7 and 5.8. Each form was purified to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme migrated to positions corresponding to apparent molecular weights of 33,000 and 75,000, respectively. The purified enzymes of pl 4.7 and 5.8 had pH optima of 8.2 and 8.5 and apparent Km values of 55.6 and 45.5 microM for PAF, respectively. Furthermore, their phospholipase C activity was significantly inhibited by the addition of 1 mM EDTA. EDTA-inactivated enzyme, however, recovered completely upon addition of Ca2+ to the original level. p-Chloromercuribenzoate markedly inhibited enzyme activity, suggesting that phospholipase C is a -SH enzyme. The physiological role of the enzyme should be evaluated, considering its specificity for a highly potent, biologically active ether-phospholipid.

Subcellular localization and some properties of intermediate-molecular-weight acid phosphatase from rat liver.

The distribution of acid phosphatases of intermediate molecular weight was determined in various rat tissues. The distribution study of intermediate-molecular-weight acid phosphatase (disignated P-II) in the subcellular fractions of rat liver and kidney indicated that P-II was localized in the mitochondrial fractions. The P-II partially purified from liver showed a pI value of 7.0 on isoelectric focusing, and the apparent molecular weight was estimated to be 40000 by Sephadex G-100 gel filtration or 44000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme catalyzed the hydrolysis of a wide variety of natural phosphomonoesters, except for phosphoproteins, phosphoserine, o-phosphocholine and thiamine monophosphate. The enzyme showed a high activity on pyridoxal phosphate, β-glycerophosphate, flavin mononucleotide and adenosine 2'-monophosphate. It was markedly inhibited by Hg2+ and Ag+, but not significantly by sulfhydryl blocking agents or by L-(+)-tartrate.

Properties of S-adenosylmethionine decarboxylase from rabbit liver: effects of some hydrazone compounds on its activity.

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been partially purified from rabbit liver by ammonium sulphate fractionation and gel filtration and anion exchange chromatographies. Sodium dodecylsulphate-polyacrylamide disc gel electrophoresis analysis showed an approximate dimeric subunit mol. wt of 34,000. The enzyme showed a pH optimum at 7.5 (in phosphate buffer) and did not require bivalent cations for catalysis. The enzyme showed sigmoid kinetics to S-adenosylmethionine with a Hill coefficient of 1.7, which became michaelian with Km 70 microM in the presence of 2.5 mM putrescine. Methylglyoxal bis(guanylhydrazone) was an effective inhibitor of the enzyme, but phenylated derivatives of this compound as phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis-(guanylhydrazone) inhibited less well.

Affinity purification of alkylglycerol monooxygenase from rat liver microsomes by chimyl alcohol-Sepharose 4B column chromatography.

Alkylglycerol monooxygenase of rat liver microsomes was purified approximately to 97-fold with a 30% yield by procedures including affinity chromatography on chimyl alcohol-Sepharose 4B. Chimyl alcohol (1-O-hexadecylglycerol) was converted to the p-aminobenzylidene derivative and then coupled to 6-carboxyhexyl-Sepharose. The final enzyme preparation was in nearly a homogeneous state, judging from the results of sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, and it migrated to a position corresponding to an apparent molecular weight of 45,000. The results revealed that the native form of the enzyme (estimated to have a molecular weight of 400,000 as judged by Sepharose 6B column chromatography in a previous report, Ishibashi, T., and Y. Imai. 1983. Eur. J. Biochem. 132: 23-27) will polymerize to large aggregates.

Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.

Toxic substance in culture filtrate of Vibrio sp. strain N7802, a poor producer of hemolysin and protease.

Culture filtrate of pathogenic Vibrio sp. (strain N7802) was fractionated by DEAE-cellulose ion exchange chromatography. The fraction showing lethal activity in rainbow trout and mice was eluted from the column with linear gradient of NaCl (DII fraction). The first peak (GI fraction) obtained by purification of DII fraction by Sephadex G-200 gel chromatography had lethal toxicity against rainbow trout and mice. Neither protease nor hemolytic activity was observed in the GI fraction. In sodium dodecylsulfate-polyacrylamide disc gel electrophoresis, the GI fraction revealed one protein band with molecular weight of 35 K and one periodic acid-Schiff positive band in the same position as the protein band. These results indicate that Vibrio sp. strain N7802 produced extracellular toxic substance which was distinct from hemolysin or protease and the toxin might play a role in the pathogenesis of vibriosis in rainbow trout.

Mode of inhibitory action of a bacteriocin produced by Streptococcus mutans C3603.

The basis for the lethal activity of a bacteriocin produced by Streptococcus mutans C3603 (serotype c) was studied. Bacteriocin C3603 was found to adsorb to cells of representative strains of the seven serotypes of S. mutans. S. mutans BHT (serotype b) was used to study the adsorption and the lethal properties of bacteriocin C3603. The adsorption of bacteriocin to cells of S. mutans BHT was inhibited by treatment of cells with protease and beta-glucosidase and by such ligands as poly-L-lysine, poly-L-arginine, L-aspartic acid, L-glutamic acid, glutathione, oxidized glutathione, poly-L-aspartic acid, and poly-L-glutamic acid. The adsorption to cells was also inhibited by oligosaccharides and glucosamine. Mixtures of anionic and cationic amino acids or polyamino acids did not greatly enhance or antagonize the inhibition of adsorption of bacteriocin C3603 to cells. Sodium hydroxide extracts of cell walls and cell wall-membranes contained carbohydrates and proteins; however, only proteins were found to bind to bacteriocin or to a bacteriocin affinity column. The sodium hydroxide extracts contained about 35 protein bands as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Bacteriocin C3603 was found to immediately inhibit the synthesis of proteins, DNA, and RNA of cells and to slowly release DNA from cells of S. mutans BHT.

Studies on the rabbit proacrosin-acrosin system

A single molecular form ( M r = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 11721–11728) was initially converted by autoactivation into an acrosin ( M r = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities ( R m = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands ( R m values of 0.395 – 0.412 and 0.497 – 0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the M r 34,000 form of acrosin. This property was not shown by M r 68,000 acrosin. Initial acrosin ( M r = 68,000) was activated by divalent cations such as Ca 2+ and Mg 2+. The enzyme was inhibited by Zn 2+, Fe 2+, Hg 2+, and sulfhydryl blockers such as 5,5′-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn 2+ is involved in acrosomal stabilization. The initial rabbit acrosin ( M r = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.

Purification and properties of two enzymatic forms of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax L. liver

Abstract 1. 1. Two isoenzymatic forms (A and B) of glucose 6-phosphate dehydrogenase ( d -glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) from bass liver have been purified to over 90% of homogeneity by gel filtration, ion exchange and affinity chromatographies. 2. 2. The apparent molecular weights were estimated by gel filtration chromatography to about 160,000 and 130,000 for form A and B, respectively. Analysis of the enzymes on sodium dodecylsulphate-polyacrylamide disc gel electrophoresis showed to be dimeric proteins. 3. 3. The effect of pH, temperature and kinetic properties for both enzymes were studied.

A functionally active heavy chain derived from human high molecular weight urokinase.

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.

Purification and partial characterization of ATP pyrophosphohydrolase from fetal bovine epiphyseal cartilage.

ATP pyrophosphohydrolase was partially purified from fetal bovine epiphyseal cartilage. The purification was about 10- and 100-fold over the enzyme activities of matrix vesicle fraction and cell homogenate, respectively. The pyrophosphohydrolase and alkaline phosphatase were separated by a sequential application of Sepharose CL-6B and DEAE-cellulose column chromatographies. The purified enzyme migrated as a single band corresponding to the molecular weight of 230,000 in sodium dodecyl sulfate-polyacrylamide disc gel by electrophoresis. The enzyme absolutely required Zn2+ for its activity and appeared to bind Zn2+ strongly with an apparent affinity of p[Zn2+]0.5 = 13.4. The apparent Km for ATP was 0.18 mM. The enzyme was also reactive toward various nucleoside triphosphates including GTP, CTP, and UTP. In contrast, various phosphodiesters including RNA, UDP-glucose, NAD, and bis-p-nitrophenylphosphate were 5% or less as reactive as the nucleoside triphosphates. The pyrophosphohydrolase was inactive toward adenosine 3':5'-monophosphate or various phosphonates. UDP-glucose (1 mM), NAD (1 mM), or RNA (1 mg/ml) failed to inhibit the ATP pyrophosphohydrolase activity. These observations suggest that the ATP pyrophosphohydrolase of the cartilage is probably not a phosphodiesterase I. The matrix vesicle fraction, which probably also included some plasma membrane vesiculated during collagenase digestion, contained the highest specific activity of the enzyme as compared to other subcellular fractions of either epiphyseal or articular cartilage.

Purification and characterization of N1-methylnicotinamide oxidases I and II separated from rat liver

N 1-Methylnicotinamide (1-CH 3Nmd) oxidases I and II of the livers of Wistar strain male rats were separated by DEAE-cellulose column chromatography and purified by AcA 34 gel filtration, Affi-Gel blue column chromatography, and sucrose density gradient centrifugation. These two enzymes were purified 568- and 82-fold with yields of 1.4 and 0.1% of the total activity in the liver homogenate, respectively. They showed the same molecular weight of 300,000 by AcA 34 gel filtration and a single protein band with a molecular weight of 150,000 in sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The difference in antigenicities of these two enzymes was observed with double immunodiffusion and immunoprecipitation using antibody produced in rabbits against each enzyme. Furthermore, these two enzymes were found to be different from each other in the heat stability, optimum pH values, K m values for 1-CH 3Nmd, and response to various inhibitors.

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin.

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.5, and pH 2.5 followed by successive gel filtrations of the pH 2.5 sperm extract supernatant over Sephadex G-75 and G-50. The resulting 8.8-fold purified materials were judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, had an estimated molecular weight of 12,800, and a constant specific activity of 65 units/mg. Treatment with the proteinases acrosin, trypsin, or chymotrypsin destroyed the highly purified proacrosin conversion inhibitor, indicating that it is a protein. Additional properties of the inhibitor included stability to long periods of storage at pH 3.0 and 4 degrees C, stability to boiling and lyophilization, and an absolute requirement for divalent cations to maintain activity. The highly purified proacrosin conversion inhibitor does not inhibit acrosin. Therefore, it apparently acts to prevent proacrosin conversion by selectively inhibiting the zymogen's self-catalyzed conversion mechanism.

Monkey pepsinogens and pepsins. Monkey pepsinogens and pepsins. V. Purification, Characterization, and amino-terminal sequence determination of crab-eating monkey pepsinogens and pepsins.

Pepsinogens were purified from the gastric mucosa of the crab-eating monkey, Macaca fascicularis. Eight pepsinogens were shown to be present disc-electrophoretically and they were termed pepsinogens I-a, I-b, III-1-a, III-1-b, III-2-a, III-2-b, III-3, and C, based on the nomenclature used for Japanese monkey pepsinogens. The molecular weights were 43,000 for pepsinogens I-a and I-b, 40,000 for pepsinogens III-1-a, III-1-b, III-2-a, III-2-b, and III-3, and 38,000 for pepsinogen C, as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Pepsinogens I-a and I-b contained carbohydrate amounting to about 4-5% by weight. Each was activated to pepsin by acidification at pH 2.0. Pepsinogen III-1 (a mixture of III-1-a and III-1-b) yielded a single pepsin, i.e. pepsin III-1, and pepsinogen III-2 (a mixture of III-2-a and III-2-b) also gave a single pepsin, i.e. pepsin III-2. The molecular weights were estimated to be 38,000 for pepsins I-a and I-b, 35,000 for pepsins III-1, III-2, and III-3, and 34,000 for pepsin C. Optimal pHs toward acid-denatured hemoglobin were 1.9, 2.3, 2.0, 2.0, and 2.3 for pepsins I-a, III-1, III-2, III-3, and C, respectively. Pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), and p-bromophenacyl bromide inhibited each pepsin. Amino acid compositions of the pepsinogens and pepsins were determined. Pepsinogen C and pepsin C were distinct from the other pepsinogens and pepsins in their high ratios of glutamic acid to aspartic acid, and leucine to isoleucine. Amino acid sequences of the amino (N)-terminal 14 residues of pepsinogens were determined by the manual Edman procedure. One to three substitutions of amino acids were observed in the 14-residue segments among the pepsinogens except for pepsinogen C. There were 7 amino acid substitutions between pepsinogens C and III-3. These results suggest that the amino acid substitutions in the N-terminal region contribute considerably to the heterogeneity of pepsinogens.

A cathepsin D-like acid proteinase from human gastric mucosa. Purification and characterization.

A cathepsin D-like acid proteinase from human gastric mucosa was purified for the first time to homogeneity as examined by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 85,000 by gel filtration on Sephadex G-150 and, after treatment with 2-mercaptoethanol, about 38,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. These results indicate that the native enzyme is composed of two apparently identical monomeric units. The protein band could also be stained with the Schiff reagent after periodate oxidation, indicating that the proteinase is a glycoprotein. Analyses showed that the enzyme contains approximately 4 glucosamine and 16 mannose residues per molecule (M.W. 85,000). The amino acid composition generally resembled those of cathepsins D except for the lower contents of basic amino acid residues, especially lysine. It also showed some similarity to pepsinogens. The optimal pH was 2.3--3.5 with hemoglobin as a substrate. The enzyme was strongly inhibited, like pepsin, by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as well as by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. The proteinase was stable in alkaline solution up to pH 9.0, and was rather stable to sequential acidification and neutralization. The acidification resulted in an increase of electrophoretic mobility towards the anode.

Preparation of antibody (IgG)-ricin A-chain conjugate and its biologic activity.

As a model experiment for the preparation of cancer chemotherapeutic agents, a disulfide-linked conjugate of rabbit anti-mouse IgG (RaMIgG) and ricin A-cahin was synthesized. Activated sulfhydryl groups were first introduced into RaMIgG and then the modified RaMIgG was reacted with ricin A-chain. The fraction containing the conjugate was obtained from the reaction mixture by gel filtration through Sephadex G-150 and was characterized by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and double immunodiffusion in agar. The inhibitory activity of A-chain against protein synthesis in a cell-free system and the binding activity of RaMIgG to mouse B lymphocytes were both fully retained. The purified conjugate selectively inhibited protein synthesis in mouse B lymphocytes possessing cell surface Ig, but not that in thymocytes. The cytotoxicity was inhibited by adding mouse IgG and an excess amount of RaMIgG to the cell culture. The effects of unlinked RaMIgG and A-chain on the cytotoxicity were negligible even when the two were used as a mixture.

Expression of Two Differentiation Antigens on Normal and Cultured Human T Cells

An antiserum specific for human T lymphocytes (AMT) was used to examine patterns of T cell surface antigen expression and to isolate their reactive membrane antigens. By a quantitative adsorption assay, different plateaus of AMT reactivity with blood T cells were observed after serial adsorptions with individual T cell lines. MOLT-3 cells removed 95% of AMT activity to blood T cells whereas MOLT-4 removed 70% and HSB-2 removed only 30%. A cross-adsorption analysis demonstrated that each of the three cell lines differed in their adsorbing efficiency to remove AMT antibodies reactive with the reciprocal cell lines. Radiolabeled membrane proteins were solubilized with either sodium deoxycholate (DOC) or NP-40 detergents, precipitated with AMT, and Staphylococcus aureus Cowan strain I, and analyzed by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis (SDS-PAGE). Two distinct T lymphocyte antigens of approximately 25,000 daltons (p25) and 16,000 (p16) were identified on MOLT-3 cells. Similar relative quantities of p25 and p16 were detected on human peripheral T cells and thymocytes. MOLT-4 cells contained less of the p25 peak than did MOLT-3. HSB-2 cells gave a small peak in the same general location as the MOLT-3 p25 peak, and a relatively large p16 peak. Adsorption of AMT with HSB-2 removed the capacity of the antiserum to precipitate p16 from MOLT-3, but did not eliminate reactivity with p25.

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.

Polypeptide Composition of Chlorophyll-Protein Complexes from Romaine Lettuce

The protein moiety of the two major chlorophyll-protein complexes associated with chloroplast membranes of outer, dark green leaves of a romaine lettuce shoot (Lactuca sativa L. var. Romana) has been analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complex II, also termed light-harvesting chlorophyll-protein complex, is shown to consist of a major polypeptide of 25 kilodaltons (kD) and two minor ones of 27.5 and 23 kD. The 25 kD subunit is the single largest polypeptide component of the chloroplast membranes, accounting for about 25% of their total protein. Complex I contains only high molecular weight subunits, the major one being at 67 kD, these subunits representing only a small percentage of the chloroplast membrane total protein.These data, suggesting an oligomeric nature for the apoprotein of these two chlorophyll-protein complexes, are difficult to reconcile with the estimated molecular weights of the native complexes and raise some intriguing questions as to the types of interactions among the components of these major lipoproteins of the photosynthetic membranes.