Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes.

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

Recombinant models of lipoproteins. Apolipoprotein A-I/phosphatidylcholine/cholesterol complexes formed in a 2-chloroethanol-water mixture

Apolipoprotein A-I can spontaneously associate with phosphatidylcholine and cholesterol in 2-chloroethanol-water mixture. It was demonstrated, using a spin label technique, that dissolved molecules participate in complex formation. The apolipoprotein A-I/phosphatidylcholine/cholesterol complexes were isolated by gel chromatography. Complexes of three types were prepared and characterized: type A, large heterogeneous aggregates with molecular weight 600 000, sedimentation coefficient 10 S and the following molar composition - protein/phosphatidylcholine/cholesterol, 1:(70-100):(10-12); types B and C, with weight average molecular weights 140 000 and 110 000, average sedimentation coefficients 3.6 S and 1.7 S, respectively. Both types have the same molar composition - protein/phosphatidylcholine/cholesterol, 1:25:8. The dissimilar sedimentation coefficients between complexes B and C may be explained by the difference in the monomer/tetramer ratio (monomer molecular weight 50 000). The spin label sn-1-O-stearoyl-2-O-9'-spiro(4',4'-dimethyloxazolidine-3'-oxyl) heptadecanoylglycero-3-phosphocholine introduced into the complexes A and B showed different thermal properties of these complexes, which may be due to differences in the lipid-protein interactions.     

An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution.

The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.     

Molecular and complex-chemical studies on methemoglobin from Chironomus thummi thummi and various isolated fractions]

1. Six different hemoglobin (Hb) fractions were isolated and characterized from the larvae of Chironomus thummi thummi using column chromatographic procedures. 2. Chromatographic and sedimentation-analytic studies (sedimentation coefficients of 2.0 +/- 0.2 (S)) have shown three Hb fractions to exist basically in a monomeric form. The molecular weight of component M-2 was determined by sedimentation equilibrium technique to be 15,470 +/- 400. The dimeric Hb was found to have sedimentation coefficients of 3.0 +/- 0.1 (S) in the weakly acidic pH region. In alkaline milieu, the reversible dissociation proceeds into the monomeric molecules (S20, W = 1.9 +/- 0.1 (S)). Molecular weights vary between pH 5.7 and 9.8 not only with hydrogen ion concentration, but also with protein concentration in correspondence with a dissociation-association equilibrium consisting of monomers and dimers. 3. For the Hb fraction M-2, a friction ratio of f/fo = 1.03 was calculated, suggesting an almost spherical shape of this protein. In contrast, the dimeric component appears to have a much more asymmetric structure (f/fo = 1.19). 4. The indivdual MetHb fractions bind the ligands: fluoride, imidazole and azide with different affinities.     

Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.     

The Fractionation of Hydrolyzed Dextran by Ultrafiltration Through A Series of Anisotropic Cellulose Acetate Membranes

Dextran, a homopolyner consisting almost solely of 1,6-α- linked glucose units, was separated into five well defined, narrow range, low molecular weight fractions by sequential ultrafiltration, after controlled lydrolysis. A commercially available purified dextran preparation, D 10, with a weight average molecular weight (Mw) of 10,980, was hydrolyzed with HCl to an average molecular weight of 4200. By ultrafiltration through a series of graded anisotropic cellulose acetate membranes of decreasing sore sizes, molecular weight fractions having Mw's of 7100, 6175, 4320, 2810 and 1565 were obtained. From Mw and sedimentation (S) values, the frictional coefficients were calculated for each fraction and the asymmetry ratios obtained therefrom.     

Natural size classes in DNA from Chinese hamster metaphase chromosomes

DNA from isolated Chinese hamster ovary (CHO) metaphase chromosomes can be obtained in three different molecular weight classes. The two largest forms have sedimentation coefficients of 80 and 120 S at 7,500 rpm. Based on sedimentation and speed dependence analysis these have molecular weights of 220 million and above 5,000 million, and are thought to be analogs of DNA classes observed in a prior study of human metaphase chromosomes. An extract can be converted to primarily the 80 S form through alkaline pH treatment of metaphase DNA. The third class (45 S DNA) is formed as a result of metaphase chromosome exposure to the nuclease Bal31, and has a mass distribution analogous to the CHO replicon.     

Identification and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by HeLa cell mitochondrial DNA

Poly(A)-containing RNA isolated from the components of a HeLa cell mitochondrial lysate which sediment in the polysome region of a sucrose gradient have been analyzed for the presence of discrete species. Eight distinct components have been identified by polyacrylamide gel electrophoresis after formaldehyde treatment. These components, which are highly reproducible in their occurrence and relative amounts under widely varying conditions of isolation, have been characterized as to their sedimentation behavior under denaturing conditions, poly(A) content and homology to separated strands of mitochondrial DNA.One of the discrete components was previously shown to have a sedimentation coefficient of about 7 S in the native state and a molecular weight of about 9.0 × 10⁴, as estimated from its sedimentation rate in formaldehyde. The molecular weights of the other seven components, as derived from sedimentation data, range between 2.6 and 5.3 × 10⁵.The 7 S RNA is complementary to the light mitochondrial DNA strand, while the other seven components are complementary to the heavy strand. Together with the two mitochondrial rRNA species and with mitochondrial 4 S RNA, the eight poly(A)-containing RNA components, if distinct in sequence, would account for about 70% of the single-strand informational content of HeLa mitochondrial DNA.     

A simple method for the determination of DNA molecular weight distribution

A method is described for the molecular weight distribution of DNA which is determined from sedimentation-velocity analysis. Knowing the distribution of sedimentation coefficients for a single DNA concentration it is possible to extrapolate such a distribution to infinite dilution of the solute in a simple way. Two versions (using two or three terms of a series) of extrapolating equations are considered and discussed in detail. The sedimentation coefficients distribution calculated from these equations differs only insignificantly with that obtained in a conventional way.     

Structural characterization of insulin receptors. I. Hydrodynamic properties of receptors from turkey erythrocytes

Insulin receptors from turkey erythrocyte plasma membranes were solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate). Their hydrodynamic properties were determined by sedimentation analyses in H2O and D2O, and gel filtration on Sepharose 4B. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peaks I and II. In Triton X-100, the sedimentation coefficient (s20,w), partial specific volume (Vc), and Stokes radius (a) for peaks I and II are, respectively, 10.2 +/- 0.5 S and 6.6 +/- 0.5 S, 0.75 +/- 0.02 ml/g, and 0.76 +/- 0.02 ml/g, and 89 +/- 3 A and 76 +/- 3 A, to yield Mr = 410,000 +/- 75,000 and 235,000 +/- 55,000, respectively, for the protein-Triton X-100 complex. The corresponding values in deoxycholate solution are: 10.7 +/- 0.5 S and 6.9 +/- 0.5 S, 0.71 +/- 0.03 ml/g and 0.70 +/- 0.04 ml/g, and 86 +/- 3 A and 69 +/- 3 A for peaks I and II, respectively, to yield 360,000 +/- 65,000 and 180,000 +/- 45,000, respectively, for the molecular weight of the protein-deoxycholate complex. These data are consistent with a model whereby each receptor species binds to one micelle of the appropriate detergent. In agreement with this model, it was also found that, in both Triton X-100 and deoxycholate, concentrations higher than the critical micellar concentration are required in order to maintain discrete receptor species in solution. At concentrations below the critical micellar concentration, the receptors aggregate to a broad band that sediments faster than 11.3 S. This is typical of membrane proteins that are stabilized in solution by insertion into detergent micelles. Based on these results, the protein molecular weights of peaks I and II are estimated to be 355,000 +/- 65,000 and 180,000 +/- 45,000, respectively. When membranes are treated with the reducing agent dithiothreitol, peak I is converted to peak II. This fact, together with the estimates obtained for the protein molecular weights of the two receptor species, suggests that peak I is a disulfide-linked dimer of peak II. The sedimentation characteristics of insulin receptors in many different cell types appear to be similar. As with turkey erythrocytes, detergent extracts of membranes from rat liver contained two native receptor species whose sedimentation coefficients were similar to peaks I and II. However, in all the other cell types examined, including rat adipocytes, rat heart muscle, 3T3-L1 adipocytes, 3T3-C2 fibroblasts, and FAO hepatoma cells, peak I (the native dimer) was the predominant species observed.     

Arylsulfatase from Pseudomonas sp. strain C12B: purification to homogeneity, immunological analysis, and physical properties.

Arylsulfatase was purified 219-fold from Pseudomonas sp. strain C12B. The final preparation was homogeneous by electrophoretic and immunological analysis. The enzyme is a monomer of molecular weight about 51,000, with a Stokes radius of 3.0 X 10(-7) cm, a frictional ratio of 1.2, and a sedimentation coefficient of 4.1S.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Analysis of Parvovirus mRNA by Sedimentation and Electrophoresis in Aqueous and Nonaqueous Solution

Adenovirus-associated virus (AAV)-specific RNA present in the cytoplasm of cells coinfected with a helper adenovirus was analyzed by sucrose gradient sedimentation and gel electrophoresis. In aqueous conditions both gels or gradients revealed three AAV RNA components corresponding to 30S, 27S, and 20S and having apparent molecular weights of 2.6 x 10(6), 1.75 x 10(6) to 1.8 x 10(6), and 0.9 x 10(6) to 1.0 x 10(6), respectively. In nonaqueous, denaturing solvents only the 20S AAV RNA species was observed. For this reason, and because they would be apparently significantly larger than a single AAV DNA strand, both the 30S and 27S species are believed to result from conformational or aggregation effects in the aqueous nondenaturing systems. It is concluded that only a single RNA molecule having a molecular weight of approximately 0.9 x 10(6) to 1.0 x 10(6) is synthesized by AAV.     

Isolation and characterization of two species of receptor which bind intrinsic factor

The intrinsic factor receptor from guinea pig ilea has been characterized following purification by affinity chromatography. The purified receptor complexed to intrinsic factor-cobalamin (holo-receptor) had an anhydrous molecular weight of 680,000, a Stokes radius of 10.9 nm, and a sedimentation coefficient of 15.1 S. In contrast, unsaturated receptor (apo-receptor) resolved into distinct "large" and "small" molecular species having, respectively, (i) molecular weights of 700,000 and 350,000; (ii) Stokes radii of 11.1 and 7.06 nm; (iii) sedimentation coefficients of 15.5 S and 11.9 S; (iv) association constants for binding the intrinsic factor-cobalamin complex of 7.3 and 2.5 X 10(10) liters mol-1; and (v) two isoproteins for the larger species (pI's 4.05 and 4.80), and one isoprotein for the smaller species (pI 4.90). A rabbit anti-receptor serum gave only one precipitation line in immunodiffusion against either the large or the small receptor, and each one of these two lines fused completely with the one of two lines which formed with the purified preparation containing both receptor species. Autoradiography of the precipitin lines obtained when the receptor was coupled to intrinsic factor-cyano[57Co]cobalamin demonstrated that both species of receptor were functional. The reaction of complete immunologic identity, the similar electrical properties and similar kinetics for binding to intrinsic factor, and the observed molecular weight differences indicate that the small and large apo-receptors are chemically interrelated, and suggest that the large receptor may consist of two small functional proteins.     

Hydrodynamic studies on interactions between the components of the liver microsomal cytochrome P-450 system

To understand the different behaviour of cytochrome P-450 systems in kinetics as well as in the demethylase activity, sedimentation and molecular weight experiments have been carried out with the following results: 1) Sedimentation coefficients of solubilized P-450 and P-450 LM2 fractions amount to 24 +/- 4 [S] and 12.8 +/- 1.2 [S], respectively. Molecular weights were determined to be 1.0 +/- 0.2 . 10(6) and 3.0 +/- 0.5 . 10(5) Dalton. 2) Triton N-101 provokes splitting of the associated structure both of solubilized P-450 and P-450 LM2; this effect is reversible. 3) The dissociation depends not only on the absolute concentration of Triton but rather on the Triton P-450 ratio. The dissociation curves of solubilized P-450 and P-450 LM2 are similar in shape and in the Triton/P-450 ratio dependence. 4) In the presence of small concentrations of Triton a more complicated dissociation behaviour was observed with broad integral distribution of the sedimentation coefficients. 5) The ionic detergent cholate splits the associated structure of P-450 LM2 at considerably higher concentrations in comparison with Triton-N 101. 6) Addition of reductase causes a decrease of sedimentation coefficients and molecular weights of solubilized P-450. The same effect in P-450 LM2 could be observed only in the presence of phospholipids.     

Further characterization of intracytoplasmic A particle-associated DNA.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.     

Heart tissue contains small and large aggregates of ferritin subunits

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.     

High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes.

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.