Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues

Class II DNA-dependent RNA polymerases were purified from soybean tissues of different physiological states: (1) from seed embryo tissue, representative of a quiescent, low metabolic state and (2) from auxin-treated hypocotyl tissue, representative of a highly proliferative and metabolically active state. Dodecyl sulfate, polyacrylamide gel electrophoresis indicates that RNA polymerase II from embryonic tissue consists largely (90-95%) of the form IIA enzyme, the largest subunit having a molecular weight of 215 000. RNA polymerase II from hypocotyl tissue is exclusively a form IIB enzyme, the largest subunit having a molecular weight of 180 000. Polypeptides common to RNA polymerases IIA and IIB have the following molecular weights: 138 000; 42 000; 27 000; 22 000; 19 000; 17 600; 17 000; 16 200; 16 100; and 14 000. Peptide mapping in the presence of dodecyl sulfate suggests that the 215 000 and 180 000 subunits possess similar peptide fragments. Plant embryo tissues do not contain protease activity capable of cleaving the 215 000 subunit to the 180 000 subunit, but proliferating plant tissues do contain such an activity. Mixing experiments indicate that appreciable amounts of RNA polymerase IIB are not being artifactually produced during protein purification.     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos

DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IIA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959-1964). Twelve polypeptides with molecular weights × 10⁻³ of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 were routinely found to be associated with the purified enzyme. From embryos germinated for 36 hours, RNAP IIB was almost exclusively obtained which has a largest subunit of 180,000 mol wt instead of 220,000. From embryos germinated for 24 hours, an approximately equimolar mixture of RNAP IIA and IIB was obtained. Peptide maps of the 220,000 and 180,000 mol wt polypeptides of RNAP IIA and IIB were virtually identical, indicative of a precursor-product relationship for the two polypeptides. In addition to these results, SDS-PAGE indicated that the stoichiometry of the 27,000 mol wt polypeptide increased at the expense of the 25,000 mol wt polypeptide during germination and concomitantly with the appearance of the 180,000 molecular weight polypeptide. No modifications (e.g. gain, loss, or altered mobilities on analytical gels) in any of the other RNAP II subunits were observed in enzyme purified from embryos after various times of germination as determined by a variety of electrophoretic analyses under denaturing conditions.     

The subunit structures of soluble and chromatin-bound RNA polymerase II from soybean

DNA-dependent RNA polymerase II is present in two forms, IIa and IIb, in germinating soybean. Form IIa is the dominant form of the enzyme in ungerminated embryos and appears to be a soluble enzyme. Form IIb increases in amount as germination progresses and is tightly bound to the chromatin template. The subunit structures of soybean RNA polymerases IIa and IIb are identical except for the molecular weights of their largest subunits which are 200,000 daltons and 170,000 daltons for IIa and IIb, respectively. The enzymes have seven common subunits: 142,000, 42,000, 26,000, 20,000, 16,000, 15,000, and 14,000 daltons.     

Characterization of the isoenzymes of pig-liver esterase. 1. Chemical Studies.

Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active-site-directed inhibitor bis(4-nitro-[14C]phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C-terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side-chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals.     

A chemical and enzymological account of the multiple forms of human liver aldehyde dehydrogenase. Implications for ethnic differences in alcohol metabolism

Three major low-pI zones of aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) may be visualized with specific histochemical staining after starch gel electrophoresis at pH 7.4 of Caucasian human liver extracts, whereas about 50% of Chinese human liver extracts show only two such zones. The three zones of activity were purified to apparent homogeneity from Caucasian liver. The substrate specificity of each form was investigated by double reciprocal plots using 13 aldehydes of various chemistries. The acetaldehyde-preferring isozyme I lacking in 50% of Chinese livers had a slightly lower native and subunit molecular weight than the "universal' isozymes IIa and IIb. All forms were highly sensitive to disulfiram inhibition. This inhibition could be protected against, or reversed, by dithiothreitol. 2,2'-Dithiodipyridine was a slower inhibitor of isoenzyme I. All three purified forms of the enzyme, as well as crude extracts of normal and isozyme I-deficient Chinese livers, showed positive immunoreactivity to antibodies prepared in rabbits against type I enzyme. Tryptic peptide maps of forms IIa and IIb were almost identical, whereas that of form I, although showing some similarities, was clearly different. These results provide a consistent explanation for the acetaldehyde-mediated extreme sensitivity to moderate alcohol ingestion shown normally by about 50% of oriental subjects and during disulfiram (Antabuse) therapy by all subjects.     

Electrophoretic microheterogeneity and subunit composition of the 13S coupling factors of oxidative and photosynthetic phosphorylation.

Two electrophoretically distinguishable species of the 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis are detectable by standard polyacrylamide gel electrophoresis in the absence of urea, detergents, or any other protein-denaturing reagents. The slower species (type IA) can be converted into the faster species (type IB) by treatment with ATP, and the fast form converts into the slow form when aged at 4 degrees. The enzyme undergoes these conversions both when it is free in solution and when it is membrane bound. The ATP analog adenylyl imidodiphosphate (AMP-PNP) gives the conversion without being hydrolyzed and without causing any apparent change in the mass of the protein, which suggests that the conversion may be a ligand-induced conformational change. Types IA and IB can convert into three other electrophoretically distinguishable species (types IIA, IIB, and III) if the purification procedure involves chromatography on a DEAE-Sephadex column equilibrated in phosphate buffer. These conversions can be prevented if the column is eluted in morpholinoethanesulfonic acid (Mes) buffer and KCl. Type IIA is convertible into type IIB by ATP treatment. Types IA and IB will also convert into types IIA and IIB and finally into type III when aged for extended periods of time at 4 degrees, without a detectable change in mass. Coupling factor activity is lost when type I enzyme converts into type II enzyme, as is the ability of the enzyme to bind to the membrane. However, ATPase activity does not change significantly. The mitochondrial 13S coupling factor shows up to three electrophoretically distinguishable species. The use of phosphate buffer during DEAE-Sephadex chromatography gives conversion of slower species into faster species. ATP treatment does not give interconversions, and aging at 4 degrees gives only a slow dissociation of the enzyme into subunits. The chloroplast 13S coupling factor also shows up to three electrophoretic species. Incubation with ATP does not give interconversions, but a temperature-dependent conversion of the major species into a faster species occurs upon aging. The subunit composition of the three 13S enzymes is very similar by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the major difference being in the number of classes of small polypeptides.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.     

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.     

Polypeptide composition of flacherie viruses of the silkworm

The purified flacherie viruses of the silkworm, Bombyx mori, (FVS I, FVS II, FVS III, and FVS IV) were iodinated by using chloramine-T. The iodinated FVSes were purified by sucrose density gradient centrifugation or 2.4% polyacrylamide gel electrophoresis. FVS IV was found in the sedimentation analysis of FVS I, FVS II, and FVS IV. Electrophoretic patterns of FVS IV showed that it was a mixture of components having identical mobilities with FVS I, EVS IIa, and FVS IIb. FVS IV was a decomposed particle of FVS I, FVS II, and/or FVS III. All of these particles contained three polypeptides with molecular weights of about 51,000, 31,000, and 12,000 daltons. FVS I composed of six polypeptides with molecular weights of 67,000, 51,000, 39,000, 31,000, 14,000, and 12,000 daltons. The maturation process of FVS I was discussed and was suggested as the following process, FVS IIb→FVS IIa→FVS I. It is not clear whether FVS III is an intermediate for FVS IIa to convert into FVS I, or FVS III is a decomposed particle of FVS I.     

Ribosomal subunit localization of hemoglobin messenger RNA.

When rabbit reticulocyte polyribosomes are treated with 0.5 M KCl, they dissociate into subunits and release a protein fraction which is required for peptide chain initiation in a cell-free system using KCl-treated subunits as the source of ribosomes. Three independent methods were used to determine the fate of mRNA after KCl treatment of the subunits. These three methods (sucrose gradient analysis of RNA after dissociating it from protein with sodium dodecylsulfate, acrylamide gel electrophoresis of RNA and electron microscopic analysis of subunits) all showed the 8--9-S mRNA to be associated with the small subunit, but not the large subunit. Furthermore, no mRNA was found to be associated with either "native" ribosomal subunit in a reticulocyte lysate.     

Determination of the sidedness of the C-terminal region of the gastric H,K-ATPase alpha subunit.

It cannot be predicted from hydropathy analysis whether the C-terminal end of the alpha subunit of the gastric H,K-ATPase is cytoplasmic or extracytoplasmic. The sideness of the C-terminal amino acids was determined by taking advantage of the two C-terminal tyrosines in the primary sequence of the enzyme. Intact, cytoplasmic side out vesicles derived from hog gastric mucosa or detergent solubilized vesicles were iodinated by the lactoperoxidase method and then the C-terminal amino acids hydrolyzed by carboxypeptidase Y. The alpha and beta subunits were separated by SDS gel electrophoresis. The level of iodination of the alpha subunit following solubilization was about three fold greater than when intact vesicles were iodinated, and the beta subunit was iodinated only when solubilized enzyme was used. Carboxypeptidase Y removed 28 +/- 4% of the radioactivity from the alpha subunit iodinated in intact vesicles. These data are consistent with a cytoplasmic location of the C-terminal amino acids of the alpha subunit and with a mostly extracytoplasmic location of the amino acids of the beta subunit.