DNA ligase changes and cell population in thymus of corticosteroid-treated chick embryo

Two successive forms of DNA ligases normally occur successively in the chicken and chick embryo thymus, a 8.2 S, before hatching and a 6.2 S, after hatching. The disappearance of the 8.2 S and the appearance of the 6.2 S together with its increased activity can be observed earlier under the effect of corticosteroids (dexamethasone (DMSO) and hydrocortisone). The biochemical, histological and cell sorting observations are consistent with the presence of the heavy enzyme in large (7.5 μm) thymocytes and the light enzyme in smaller (5 μm) T-antigen possessing cells. These results are discussed on the basis of the effect of steroids on thymocyte maturation and with regard to cell migration within the lymphoid system.     

NAD+ kinase: molecular weight determination by low-angle laser-light scattering

Low-angle laser-light scattering (LALLS) was employed to measure the absolute molecular weight of chicken liver NAD+ kinase (NADK). The weight-average molecular weight (Mw) was found to be 275 000 +/- 15 000. The corresponding value for the second virial coefficient was -1.65 X 10(-3) ml X mol X g2. The value for Mw is in close accord with estimates reported for pigeon liver (270 000) and C. utilis (260 000) NADK. If the active enzyme is a dimer, the weight difference between pigeon/chicken liver and rabbit liver (136 000) NADK would indicate that the latter enzyme is an active monomer unit.     

Immunoglobulin G biosynthesis in a human lymphoblastoid cell line. Differences between membrane-bound and secretory forms of gamma chains

The cultured human B lymphoblastoid cell line Maja synthesises two forms of the gamma heavy chain of immunoglobulin G (IgG) that differ in apparent molecular weight. The lower-molecular-weight form is secreted into the culture medium as a water-soluble product in association with light chains and comigrates on dodecyl sulphate polyacrylamide gels with serum IgG gamma chains. The higher-molecular-weight form is not detected in culture supernatants. In distinction to the secreted form, the higher-molecular-weight form is labelled by a lipophilic, photoactivatable nitrene and is inserted asymmetrically in a transmembrane orientation into rough microsomes. It is concluded that Maja cells synthesise secretory (gamma s) and membrane-associated (gamma m) forms of IgG heavy chains. Both forms of the gamma heavy chain are glycosylated, and can contain one or two asparagine-linked glycan units. The gamma m and gamma s heavy chains differ by about 10 000 in apparent molecular weight. This difference resides exclusively in the polypeptide moiety. Although part of the difference comprises a transmembrane peptide and a cytoplasmic tail of apparent molecular weight about 2000 for gamma m chains, a substantial segment of unique peptide is most probably present on the non-cytoplasmic side of the bilayer. The ionophore monensin inhibits the intracellular transport of gamma s and gamma m chains at a stage when they are sensitive to the enzyme endo-beta-N-acetylglucosaminidase H. In contrast, HLA-A and HLA-B antigens reach a stage at which they are insensitive to this enzyme in the presence of monensin, although their surface expression is inhibited by the ionophore. The implications of these results for the intracellular transport of membrane-associated glycoproteins are discussed.     

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.     

Isolation of NAD-kinase from pigeon heart

A procedure of isolation of pigeon breast muscle NAD-kinase resulting in a 100--130-fold purification of the enzyme with a total yield of 30--35% is described. The enzyme is electrophoretically homogenous; its molecular weight as determined by SDS-electrophoresis is 45 000. The partially purified preparation contains multiple enzymic forms with molecular weights varying from 45 000 to 270 000, which represent an equilibrious system of structurally different oligomers. At the last purification step, i. e. ion-exchange chromatography, the enzyme loses its ability for oligomerization. Possible causes of disappearance of the enzyme multiple forms during purification are discussed.     

Regulatory properties of 14 day embryo and adult hen heart AMP-deaminase

Chromatography on phosphocellulose column revealed changes in the elution profile of chicken heart AMP-deaminase during ontogenesis. The extracts from the heart of adult hen and 14 day-old embryo displayed a single peak of the enzyme activity at a slightly different elution volume, whereas in the heart extract of 1 day-old chicken two molecular forms of adenylate deaminase have been eluted. The kinetic and regulatory properties of the purified adult hen heart AMP-deaminase were studied and compared with those of the corresponding enzyme from 14 day-old embryo heart. Both enzymes exhibited a slightly sigmoid-shaped plot of the reaction rate versus substrate concentration, which shifted to hyperbolic form when ATP or ADP were added into the incubation medium. The enzymes were strongly activated by ATP, less efficiently by ADP and the activatory effect was enhanced at low substrate concentration. Orthophosphate inhibited both enzymes but this inhibition was more potent for the embryo heart enzyme. Palmitoyl-CoA inhibited adult hen but not the embryo heart AMP-deaminase. The data presented indicate that the differences also in the regulatory properties of the molecular forms studied do exist and correspond with the ontogenetic differences observed previously (Kaletha and Skladanowski (1981) Experientia 37, 232-234) concerning the effect of temperature on the chicken heart adenylate deaminase.     

Chicken liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular properties.

A homogeneous amidophosphoribosyltransferase (EC 2.4.2.14) preparation, which was sensitive to purine nucleotide inhibitors, was obtained from chicken liver. From the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the subunit weight was estimated to be approximately 58 000. In Tris-HCl buffer, the predominant form of the enzyme had an S20,w of 6.5, Strokes radius of 40 A, and estimated molecular weight of 110 000. Incubation with 5-phosphoribosyl 1-pyrophosphate or Pi resulted in an increase in the S20,w to 9.1--9.5, Strokes radius 50 A, and estimated molecular weight to 200 000. Incubation of the large form with AMP led to a decrease in the molecular wight of the enzyme. It is concluded that chicken liver amidophosphoribosyltransferase is an allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Quaternary structure of NAD-kinase from rabbit liver

A procedure for isolation of NAD-kinase from rabbit liver resulting in 4000-fold purification and the activity yield of 50-60% is described. The molecular weight of the NAD-kinase subunit determined by SDS electrophoresis is 30 000. The purified enzyme is a dimer. Partially purified preparations of NAD kinase contain multiple forms with mol. Weights ranging from 650 000 to 180 000 and have complex kinetic behaviour. A thermostable activator of NAD-kinase which, when added to the homogeneous enzyme preparation, destroys the linear dependence of the enzyme specific activity on concentration, was detected. The nature of multiple forms of NAD-kinase and the possible role of the activator in their formation is discussed.     

Purification and isoelectric heterogeneity of chicken tyrosinase

Tyrosinase (EC 1.14.18.1) was purified from regenerating chicken feathers. Most of the enzyme activity was in the insoluble fraction, which was solubilized with 0.5% sodium cholate. Solubilized tyrosinase showed multiple forms on isoelectric focusing. The isoelectric points had the following pI values: 5.06, 4.83, 4.68, 4.56, 4.44, 4.32, 4.24, 4.14, 4.06 and 3.97. This tyrosinase fraction was subjected to trypsin (EC 3.4.21.4) cleavage, Sephacryl S-200, hydroxylapatite and DEAE-cellulose chromatography. Purified enzymatically active tyrosinase also showed multiple forms. Their isoelectric points were: 4.23, 4.14, 4.06, 3.99 and 3.91. Each active form had almost the same molecular weight, estimated at 66 000. Staining for 1,2-diol groups of glycoproteins and neuraminidase (EC 3.2.1.18) treatment suggested that chicken tyrosinase is a glycoprotein. The enzyme showed both dopa(L-3,4-dihydroxylphenylalanine) oxidase activity and tyrosine hydroxylase activity.     

The characterization of multiple forms of kynurenine formidase in Drosophila melanogaster.

Two enzymic forms of kynurenine formamidase (EC 3.5.1.9) from Drosophila melanogaster were separated and partially purified by pH fractionation, (NH4) 2SO4 fractionation and Sephadex G-75 gel filtration. The enzymes were also separated by DEAE-cellulose ion-exchange chromatography and distinguished by their different rates of thermal inactivation. The multiple forms are termed formamidase I and formamidase II. The molecular weight of formamidase I as measured by Sephadex G-75 chromatography is 60 000 and that of formamidase II is 31 000. The pH optima are broad, ranging between 6.7 and 7.8 for formamidase I and 6.5 and 8.0 for formamidase II. The apparent Km values are 5-10(-3) and 0.83-10(-3) M, resepctively. The possibility that formamidase II is an active subunit of formamidase I is discussed, although neither enzyme will convert to the other when separated and rechromatographed. Eight organisms were tested for the presence or absence of multiple forms of formamidase. Drosophila melanogaster and Drosophila virilis have both enzymes; cow, chicken, yeast and housefly have formamidase I only, and mouse and frog have formamidase II only.     

Identification of an active site histidine in urokinase.

Two forms of urokinase (EC 3.4.99.26) with apparent molecular weights of 33 400 and 47 000 purified by affinity chromatography have been modified specifically with newly synthesized peptide chloroketones by affinity labeline. Rapid inactivation of the enzyme preparations was observed with Ac-Gly-Lys-CH2 Cl and Nle-Gly-Lys-CH2 Cl which might be associated with a change in which a histidine residue is lost. After performic acid oxidation, an equivalent amount of 3-carboxymethyl histidine could be recovered, indicating alkylation at the N-3 of a histidine residue. In the case of the norleucine derivative, norleucine was concomitantly incorporated into the protein. It is thus likely that urokinase belongs in the class of enzymes utilizing the Asp..His..Ser triad for their catalytic action. The two active site residues so far identified, serine and histidine, were located in the heavy chain (33 100 mol. wt) of the 47 000 molecular weight form and in the 33 400 molecular weight form, the molecular weight of which remained constant.     

In vitro and in vivo age-related modification of human erythrocyte phosphoribosyl pyrophosphate synthetase.

Upon storage, human erythrocyte phosphoribosyl pyrophosphate synthetase (PRibPP synthetase, EC 2.7.6.1) from normal individuals was found to undergo a spontaneous dissociation into active enzyme components of much smaller molecular mass (60 000--90 000). These modified forms of enzyme exhibit kinetic properties different from the original large molecular weight enzyme (over 200 000). The small active components can be reversibly associated to form larger molecules in the presence of purine ribonucleotides as well as phosphoribosyl pyrophosphate (PRibPP). ATP was found to be most effective in associating PRibPP synthetase, while guanylate nucleotides seem to have no effect. The large molecular weight components, once separated from the milieu, were not able to undergo further dissociation. Fresh or stored human white cell tissue homogenates were found to lack the low-molecular-weight enzyme under all our experimental conditions. A characteristic enzyme modification similar to that observed in stored erythrocyte was also noted in erythrocytes of increasing ages. The physiological significance of these findings to the regulatory function of PRibPP synthetase in purine metabolism in vivo is discussed.     

The isolation and characterization of sphingomyelinase from human placental tissue.

Human placental sphingomyelinase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 000-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.     

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.     

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

Isolation, some properties and heterogeneous nature of DNA-topoisomerase (relaxing enzyme) from Ehrlich ascites carcinoma cells

DNA-topoisomerase catalyzing the conversion of a superhelical circular covalently closed DNA molecule into a super-helix free circular molecule, was isolated from mouse Ehrlich ascites carcinoma cells and purified 209-fold. The optimal conditions for the action and stability of the enzyme were elaborated. Using polyacrylamide gel electrophoresis under non-denaturating conditions as well as in the presence of Na-DS the heterogeneity of purified DNA-topoisomerase was established. This heterogeneity implies the presence of three active forms of the enzyme with Mr of 97 000, 81 000 and 69 000, respectively. Using one-dimensional fingerprint method and limited proteolysis with Staphylococcus aureus protease, it was demonstrated that the low molecular weight enzyme forms are products of limited proteolysis of the highest molecular weight form of DNA-topoisomerase.     

Properties and subunit composition of RNA polymerase II from plant cell cultures.

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.     

Multiple molecular forms of glia maturation factor.

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.