Studies of the subunit structure of wheat germ ribonucleic acid polymerase II

We have previously presented a rapid, high yield method for the large scale purification of homogeneous RNA polymerase II from wheat germ (Jendrisak, J.J., and Burgess, R.R.(1975), Biochemistry 14, 4639), and we now report a detailed study of its subunit structure. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that polypeptides with molecular weights of 220 000, 140 000, 40 000, 27 000, 25 000, 21 000, 20 000, 17 800, 17 000, 16 500, 16 000, and approximately 14 000 are associated with the enzyme. Two-dimensional polyacrylamide gel electrophoresis, by which the subunits were separated in the first dimension in the presence of 8 M urea at pH 8.7 and in the second dimension in the presence of 0.1% sodium dodecyl sulfate, indicates that the 40 000 molecular weight component is composed of two nearly identical polypeptides and that the low molecular weight components (smaller than or equal to 40 000) are acidic proteins except for the 25 000 molecular weight polypeptide.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Evaluation of environmental polychlorobiphenyls and DDE in terms of mixtures of commercial preparations from peak heights of packed-column gas chromatograms using a programmable calculator

Various photochemical activities were tested on chloroplasts of Zea mays that received 4 s of light every 4 h during the culture period. Photosystem I and Photosystem II were functioning, as well as the photosynthetic electron transport. These chloroplasts exhibited upon sodium dodecyl sulphate gel electrophoresis neither Complex 1 (Mr 70 000) generally associated with Photosystem I nor Complex 2 Mr 25 000) generally associated with Photosystem II. Chlorophyll is indeed attached to polypeptides of molecular weight 21 000 and 29 000. These results lead us to question the functional role of chloroplast protein-pigment complexes observed by sodium dodecyl sulphate gel electrophoresis.     

Chemical and physical properties of the human urinary glycoprotein with gastric antisecretory activity

The chemical and physical properties of the high-molecular-weight glycoprotein (SO20, w = 8S; Ve=Vo on Sephadex G-200) with gastric antisecretory activity extracted from the urine of pregnant women were studied. Gel filtration in the presence of sodium dodecyl sulphate and sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis indicated subunit mol.wts. of 16 000 +/- 1500 and 13 000 +/- 1000 respectively. Reaggregation of the subunits and partial recovery of the biological activity were observed on removal of the detergent. The partial C-terminal sequence was found to be Phe-Tyr-Leu-Val-OH, whereas glycine appears to be the N-terminal amino acid. The carbohydrate composition was examined; all galactosamine was found to be O-glycosidically linked to the polypeptide chain.     

Chromatin-bound protease: (3H)diisopropyl fluorophosphate labeling patterns of chromatin

The nuclei and chromatin of rat liver contain three major proteins reacting with diisopropyl fluorophosphate (DFP). The molecular weights of the three proteins determined by gel filtration in the presence of sodium dodecyl sulfate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 70000, 60000, and 25000. The chromatin isolated from whole liver, instead of nuclei, contains an additional DFP-binding protein whose molecular weight is 100000 in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. The small molecular weight DFP-binding protein can be fractionated from chromatin by 0.25 N HC1 and was found to be a protease which is active in the most commonly used solution for chromatin dissociation, that is, 2-3 M NaCl-5 M urea. This enzyme appears to be the major DFP-binding chromatin-bound protease in the chromatin of most rat tissues. The acid-soluble protease is converted from a 25000-dalton form to a 20000-dalton form during 0.25 N HC1 acid extraction from chromatin, which retains proteolytic activity.     

Affinity purification and some molecular properties of human liver alkaline phosphatase

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

The immunogenic effect of purified antigens of B. alexandrina against Schistosoma mansoni in experimental animals (I) as measured by worm load and viability of ova

Current stratigies for the control of schistosomiasis are based primarily on chemotherapy but successful vaccination against infection has been also demonstrated in several host parasite models. In this study, the immunogenic effect of two purified antigens (Fiv 20-29 000 daltons & Fv 20-24 000 daltons) extracted from non infected hepatopancreas of B. alexandrina as measured by worm load, state of copulation and viability of ova in tissues. The antigens were prepared using gel filtration chromatography and their molecular weights were estimated through sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four dilutions from each antigen were prepared and injected in two groups (33 each) of Swiss albino mice at weekly intervals. A control group was injected with phosphate buffer saline (PBS) in the same manner. All mice were injected with 100 cercariae using immersion method. Sacrification was done regularly after 7 weeks of injection. On the basis of worm load, Fiv gave protection of 44% while Fv gave only 36%. The number of worms in copula was significantly reduced and they became delicate, fragile, stunted and malformed. Both antigens gave a significant reduction in total viable eggs in tissues.     

Renal renin output during continuous intracarotid infusions of iso- and hypertonic sodium chloride solutions in the rat

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.     

Comparative studies of human and chicken retinol-binding proteins and prealbumins

Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.     

Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization

The complete purification of malate dehydrogenase (EC 1.1.1.37) from extremely halophilic bacteria of the Dead Sea is described. The purification procedure includes (a) precipitation by ammonium sulfate, (b) fractionation on Sepharose 4B using a decreasing concentration gradient of ammonium sulfate, (c) gel permeation chromatography on Sephadex G-100, (d) chromatography on hydroxylapatite, and (e) affinity chromatography on 8-(6-aminohexyl)amino-NAD+-Sepharose at 4.26 M NaCl. The absorption and fluorescence spectra of the native and denatured enzyme were measured, and the extinction coefficient at 280 nm in 4.26 M NaCl was found to be 0.803 cm2mg-1. The amino acid composition analysis showed an excess of 10.4 mol % of acidic amino acids. The apparent specific "volume" phi' of the active enzyme at 4.26 M NaCl was found to be 0.680 +/- 0.015 mL/g. The molecular weight of the native enzyme was found to be 84 000 +/- 4000 determined in 4.26 M NaCl from equilibrium sedimentation data. The molecular weight of the subunits is 39 000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the native enzyme is composed of two subunits.     

Characterization of high- and low-molecular weight zinc-dependent acid phosphatases in bovine liver

We have purified two forms of Zn2+-dependent acid phosphatase (Zn2+-APase) from bovine liver, both of which require Zn2+ to hydrolyze the substrate p-nitrophenyl phosphate in an acidic environment. The apparent molecular weights of these two forms of Zn2+-APase were estimated to be about 100,000 and 62,000 by gel filtration, and about 44,000 and 31,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The low-molecular weight (LMW) Zn2+-APase catalyzed the hydrolysis of myo-inositol-1-phosphate in the presence of 3 mM Mg2+ at physiological pH, but the high-molecular weight (HMW) enzyme did not. The LMW-Zn2+-APase of bovine liver was recognized by polyclonal antibodies developed against the Zn2+-APase of bovine brain, but the HMW-Zn2+-APase was not.     

Studies on almond emulsin beta-D-glucosidase. I. Isolation and characterization of a bifunctional isozyme

A beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme has been isolated from almond emulsin. The isolated enzyme is a glycoprotein and migrates as a single band on Sephadex G-200 filtration, CM 52 ion exchange chromatography, polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focussing. The glucosidase and galactosidase activities traverse together during Sephadex G-200 gel filtration. Polyacrylamide gels stained specifically for the 2 enzymes reveal that the two activities comigrate. The molecular weight of the isozyme has been found to be 135 180 +/- 770, and that of its protomers to be 65 150 +/- 650.     

Non-histone chromatin proteins during maturation of avian erythroid cells

1. In chicken erythroid cells (erythroblast, reticulocyte and erythrocyte) maturation was accompanied by a decrease in the content of non-histone chromatin proteins. 2. Phenol-soluble non-histone chromatin proteins (phosphoproteins) from the three cell populations studied, showed differences in the behavior on sodium dodecyl sulphate polyacrylamide-gel electrophoresis, and isoelectric focusing. Phosphoprotein of immature cells had a higher content of fractions of about 86 000 and 23 000 daltons than the phosphoproteins of erythrocytes.     

Further studies on bilitranslocase, a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, alpha and beta , of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is alpha 2-beta.     

Interactions of the components of the prothrombinase complex

1. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) of house-fly head tissue was solubilized as a 7.4-S form by autolysis for 80-100 h at 25 degrees C and pH 8.0. 2. The autolysed enzyme was purified by affinity chromatography, firstly on Con-A-Sepharose and subsequently on m-trimethylammoniumaniline-Affi-Gel 202. This sequence permitted overall purification yields of approx. 50% of the solubilized enzyme. 3. The 7.4-S purified enzyme was essentially homogeneous on polyacrylamide gel electrophoresis, and its specific activity coincided with the highest previously reported for fly-head acetylcholinesterase. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and beta-mercaptoethanol revealed two major polypeptide components of molecular weight 82 000 and 59 000. Each of these polypeptides contained diisopropylphosphofluoridate-binding sites, as shown with [3H] diisopropylphosphofluoridate. 5. The results suggest a strong structural similarity between fly-head acetylcholinesterase and the purified electric eel enzyme.     

Rat intestinal brush border membrane peptidases. I. Solubilization, purification, and physicochemical properties of two different forms of the enzyme

Two brush border peptidases have been isolated from the particulate fraction of the rat intestinal mucosa and purified to homogeneity as judged by polyacrylamide gel electrophoresis, starch gel electrophoresis, isoelectric focusing, and double immunodiffusion. For convenience, the peptidases have been designated peptidase F (fast) and S (slow) on the basis of their anodic mobilities. The isoelectric point of peptidase F was 4.76 and of peptidase S, 5.10. Both enzymes are glycoproteins. The amino acid compositions of the two peptidases are similar. The same carbohydrates are found in both enzymes, but there are differences in the molar concentrations of individual sugars. Peptidase S has greater concentrations of mannose and galactose and of hexosamines than peptidase F, while sialic acid is slightly greater in peptidase F. Carbohydrate accounted for approximately 19% and 23% of the weight of peptidases F and S, respectively. Estimates of the molecular weights of both enzymes by gel filtration gave values of 280,000. Electrophoresis of the enzymes under denaturing conditions on sodium dodecyl sulfate polyacrylamide gels indicated that each enzyme is a dimer consisting of two subunits of equal molecular weight, 140,000.     

Boar proacrosin. Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm

Two forms of proacrosin have been purified from ejacualted boar spermatozoa. The isolation method utilized benzamidine to inhibit the premeture activation of the zymogen and included pH precipitation, ammonium sulfate fractionation, and sodium chloride precipitation. Further purification was achieved by Sephadex G-200 FILTRATION OF THE PREPARATION AFTER IT WAS TREATED WITH 8 M urea. The overall proacrosin yield was 58% with a specific acitivity of 253 units/mg of protein. The molecular weights of the proacrosins determined by sodium dodecyl sulfate disc gel electrophoresis were 55,000 and 53,000. Proacrosin autoactivation followed the classical S-shaped activation curve and calcium was not required to obtain full activation. Time course activation studies in 0.1 M Tris/HCl, pH 8.4, at 0 degrees were monitored with sodium dodecyl sulfate-disc gel eletrophoresis and anlytical gel electrophoresis with staining techniques for protein and enzymatic activity. Under the conditions used, the zymogens were sequentially degraded to three different active specise of acrosin (alpha, beta, and gamma). The approximate molecualr weights of the acrosins were 49,000, 39,000, and 25,000 for the alpha, beta, and gamma forms, respectively. The autoactivation is concentration-dependent and can be proteolytically stimulated with either alpha- or beta-acrosin and trypsin, indicating the activation of proacrosin can via a bimolecular process.     

Subclinical vitamin A deficiency in undersix children in Nagpur, India

The CP-I subunit of calf kidney adenosine deaminase complexing protein (ADCP), isolated by affinity chromatography based on Sepharose-4B immobilized adenosine deaminase, is identical with dipeptidyl peptidase IV. This finding is based on the following results: (a) Its M(r) = 110 kD, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis; (b) its catalytic activity toward Gly-Pro-p-nitroanilide; (c) its inhibition by serine protease inhibitor; and (d) by two peptide sequences resulting from its trypsin proteolysis. Accordingly, the CP-I subunit of ADCP isolated from bovine kidney is DPPIV (CD26). Thus, as anticipated, the high affinity between ADA subunits prevails even when they originate in different species.     

Improved chromatographic purification of pea seedlings diamine oxidase

An improved and simplified purification procedure has been developed for the isolation of diamine oxidase from pea seedlings (DAO EC 1.4.3.6). It involves ammonium sulphate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography and size-exclusion chromatography. The homogeneity of the final enzyme preparations and molecular weight were determined by size-exclusion chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS PAGE). The isoelectric point of 7.35 +/- 0.05 was determined by chromatofocusing and by polyacrylamide gel isoelectric focusing.     

Identification and characterization of differentiating soluble antigens of sheep and goat poxviruses

Soluble antigens of sheep and goat poxviruses (SPV, GPV) were isolated and purified from scab suspensions prepared from lesions of experimentally infected homologous hosts. The soluble antigens were then subjected to sequential ammonium sulphate precipitation. All the obtained fractions reacted in counter immunoelectrophoresis (CIE) with both the antisera against SPV and GPV except the fraction obtained at 30% saturation level (30% SSPV), which did not react with antiserum against GPV. This differentiating soluble SPV antigen was found to consist of 210 K proteins in exclusion chromatography. The 210 K proteins contained 3 polypeptides of 100, 35 and 17 K in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). The study thus gave an evidence that the SPV-specific proteins are of a higher molecular mass nature.