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1.
Studies on the immunological cross-reactivity and physical properties of fatty acid synthetases 总被引:6,自引:0,他引:6
S Smith 《Archives of biochemistry and biophysics》1973,156(2):751-758
Fatty acid synthetase enzymes were purified from the liver, mammary gland, and adipose tissue of rats and the liver and mammary gland of mice. The enzymes from the liver and mammary gland of the same species have similar molecular weights and and dissociate into subunits at comparable rates.Rabbit antisera were prepared against the fatty acid synthetase from the lactating rat mammary gland. Cross-reactivity between different fatty acid synthetases was determined by immunodiffusion and immunoprecipitin tests. No differences in immunological cross-reactivity could be detected in liver, mammary gland, and adipose enzymes from the same species; fatty acid synthetases from the rat and mouse gave reactions of incomplete identity. Partially purified fatty acid synthetases from pigeon liver and rabbit mammary gland did not react with the antiserum.It is concluded that the immunochemical approach is useful in determining the degree of resemblance between fatty acid synthetases from different species. Within a given species, the liver and mammary gland fatty acid synthetases seem to be very similar, if not identical, proteins. 相似文献
2.
Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.
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J Knudsen 《The Biochemical journal》1979,181(2):267-274
The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue. 相似文献
3.
The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes. 相似文献
4.
Pathogenic strains of Pseudomonas glycinea were shown to possess plasmid deoxyribonucleic acid by dye-buoyant density gradient centrifugation. The size and number of plasmids of four different isolates were determined by neutral sucrose gradient centrifugation. Two isolates were found to harbor a single plasmid; however, they differed in size, having molecular weights of 43 X 10(6) and 54 X 10(6). Two other isolates each contained two different plasmids. Plasmids with molecular weights of 43 X 10(6) and 73 X 10(6) were observed in one isolate, and the other carried plasmids with molecular weights of 25 X 10(6) and 87 X 10(6). An auxotrophic mutant derived from the latter strain was found to contain plasmids of identical size. The plasmids were found to be under stringent control of replication, having plasmid copies of 1.0 to 2.7 per chromosome equivalent. By the dye-cesium chloride technique, the mutant showed twice as much covalently closed circular deoxyribonucleic acid as did the parental strain. 相似文献
5.
A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E. coli MRE-600 was developed. It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite). The mode of cell destroying was found to affect the process of the further enzyme purification. The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein. The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG. The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha). The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000. The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively. 相似文献
6.
Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy. 总被引:11,自引:1,他引:10
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The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments. 相似文献
7.
Henryk Berbeć Alicja Paszkowska Tomasz Borkowski 《Molecular and cellular biochemistry》1984,62(2):149-155
Summary Aminoacyl-tRNA synthetase activity for alanine, glutamic acid, lysine and phenylalanine was studied in the three subcellular fractions of rabbit liver: fraction X, microsomes and cytosol. From 60 to 80% of the enzyme activities were found in fraction X and microsomes. Fraction X was especially rich in the synthetase activities. By means of gel chromatography, heavy (over 106 daltons) and light (below 480 × 103 daltons) forms of lysyl- and phenylalanyl- but only light ones of alanyl- and glutamyl-tRNA synthetase activities were found in all the subcellular fractions studied. It is concluded that in higher organisms (mammals) all aminoacyl-tRNA synthetases, at least in part, are associated with cell structural constituents.Abbreviations ALA, GLU, LYS, PHE
alanyl-, glutamyl-, lysyl-, phenylalanyl-tRNA synthetase
- PMSF
phenylmethylsulfonyl fluoride
- BSA
bovine serum albumin 相似文献
8.
Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems of various microorganisms and animal tissues. This antibiotic specifically blocks the activity of beta-ketoacyl thioester synthetase (condensing enzyme) by binding to the functional cysteine-SH in the active center of the condensing enzyme domain (the peripheral SH-group). However, fatty acid synthetase from C. caerulens is much less sensitive to cerulenin than fatty acid synthetases from other sources. The properties of C. caerulens synthetase were investigated and compared to those of Saccharomyces cerevisiae synthetase, which is sensitive to the antibiotic. The molecular weight of the enzymically active form of C. caerulens synthetase was 2.53 X 10(6). The enzyme consisted of two multifunctional proteins, alpha and beta, which are arranged in a complex, alpha 6 beta 6. The synthetase was inactivated by iodoacetamide. At 0 degrees C and pH 7.15, the second-order rate constant of k = 15.6 M-1 X s-1 was obtained for the inactivation by iodoacetamide. This value was about 15 times greater than that for S. cerevisiae synthetase. Treatment of C. caerulens synthetase with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity. When S. cerevisiae synthetase was preincubated with cerulenin, malonyl-CoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide (Kawaguchi, A., Tomoda, H., Nozoe, S., Omura, S., & Okuda, S. (1982) J. Biochem. 92, 7-12). In the case of C. caerulens synthetase, on the other hand, malonyl-CoA decarboxylase activity was induced by iodoacetamide even after the preincubation of the enzyme with cerulenin.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl-CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250 000-Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185-193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10 400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847-Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins. 相似文献
10.
Molecular weight determinations of native and subunit RNAs of murine mammary tumor virus (MuMTV), a type B oncornavirus, were performed by polyacrylamide gel electrophoresis and compared with molecular weights of well-characterized avian cellular RNAs and tobacco mosaic virus RNA. From extrapolations of semilog plots of the molecular weights of the standard RNAs versus relative electrophoretic mobilities and Ferguson plots, the subunit and native RNAs of MuMTV were found to possess molecular weights of 2.93 X 10(6) and 6.45 X 10(6), respectively. These data support the assumption that two subunit molecules comprise the native RNA of MuMTV. 相似文献
11.
The effect of beta-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ. 总被引:1,自引:1,他引:0
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1. The rate of mammary-gland lipogenesis measured in vivo from 3H2O was suppressed after decreasing the milk demand by decreasing the number of pups from ten to two or three, as well as by giving diets containing lipid [Grigor & Warren (1980) Biochem. J. 188, 61-65]. 2. The specific activities of the lipogenic enzymes fatty acid synthase, glucose 6-phosphate dehydrogenase and 'malic' enzyme increased between 6- and 10-fold in the mammary gland and between 2- and 3-fold in the livers during the first 10 days of lactation. The increases in specific activity coupled with the doubling of liver mass which occurred during pregnancy and lactation resulted in considerable differences in total liver activities when compared with virgin animals. 3. Although consumption of a diet containing 20% peanut oil suppressed the activities of the three lipogenic enzymes in the livers, only the 'malic' enzyme was affected in the mammary glands. 4. In contrast, decreased milk demand did not affect the specific activities of any of the liver enzymes, whereas it resulted in suppression of all three lipogenic enzymes of the mammary glands. There was no effect on either the cytoplasmic malate dehydrogenase or the lactate dehydrogenase of the mammary gland. 5. In all the experiments performed, the activity of the fatty acid synthase correlated with the amount of material precipitated by the rabbit antibody raised against rat fatty acid synthase. 相似文献
12.
Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form. 相似文献
13.
T Koide 《Journal of biochemistry》1979,86(6):1841-1850
1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively. 相似文献
14.
Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1). 相似文献
15.
Purification and properties of a thioesterase from lactating rat mammary gland which modifies the product specificity of fatty acid synthetase 总被引:12,自引:0,他引:12
An acyl coenzyme A hydrolase (thioesterase II) has been purified to near homogeneity from lactating rat mammary gland. The enzyme is a monomer of molecular weight 33,000 and contains a single active site residue. The enzyme is specific for acyl groups, as acyl-CoA thioesters, containing eight or more carbon atoms and can also hydrolyze oxygen esters. Thioesterase II is capable of shifting the product specificity of rat mammary gland fatty acid synthetase from predominately long chain fatty acids (C14, C16, and C18) to mainly medium chain fatty acids (C8, C10, and C12). Thioesterase II can restore the capacity for fatty acid synthesis to fatty acid synthetase in which the thioesterase component (thioesterase I) has been inactivated with phenylmethanesulfonyl fluoride or removed by trypsinization. No evidence was found of significant levels of thioesterase II in lactating rat liver. The presence of thioesterase II in the lactating mammary gland and the ability of the enzyme to hydrolyze acyl-fatty acid synthetase thioesters of intermediate chain length, are indicative of a major role for this enzyme in the synthesis of the medium chain fatty acids characteristic of milk fat. 相似文献
16.
Characterization of a unique enzyme complex composed of S-adenosyl-L-methionine-tRNA-methyltransferase and aminoacyl-tRNA synthetase activities 总被引:1,自引:0,他引:1
S-adenosyl-L-methionine-tRNA methyltransferases of a murine leukemia cell line were found to exist in a high molecular weight enzyme complex. Aminoacyl-tRNA synthetase activity always co-chromatographed and co-sedimented with methyltransferase activity in evidence of a unique association of these two groups of enzymes. Molecular weight studies showed a probable molecular weight of 9 X 10(5) daltsons for the intact complex which dissociates to complexes of 6 X 10(5) and 3 X 10(5) daltons. The complexes contain discrete polypeptides of 25,000-90,000 daltons as determined from SDS-gel electrophoresis. High resolution fatty acid analysis showed that only very small amounts of saponifiable lipids were associated with the purified enzyme complex. Similarly very little protein-bound sugars was found within the complex indicating that neither lipids nor sugars were involved in the protein-protein interactions of the complex. Analysis of tRNA methylated in vitro indicated the presence of most methyltransferase activities in the purified complex. Of note was the absence from the complex of the methyltransferase responsible for the production of ribo Tp. 相似文献
17.
The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. 总被引:1,自引:0,他引:1
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The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found. 相似文献
18.
O I Gudzera A V El'skaia G V Ovcharenko L L Ivanov I D Baturina 《Molekuliarnaia biologiia》1979,13(3):550-558
Three forms (E1, E2 and E3) of leucyl-tRNA synthetase (LeuRS) were separated by DEAE-cellulose chromatography of total aminoacyl-tRNA synthetases from cow lactating mammary gland. The method of purification of all three components is described. E1 is a dimeric molecule (alpha 2) of molecular weight 182 000. Two other forms of molecular weight 67 000 and 64,000 consist of a single polypeptide chain as determined by polyacrylamide gel electrophoresis. Optimum conditions and kinetic parameters of leucyl-tRNA formation were studied for every enzyme form. The low values of Vmax and thermostability are characteristic of E3. All forms of LeuRS interact with 6 isoaccepting tRNA(Leu) from lactating mammary gland and can activate leucine in the absence of tRNA. E2 and E3 are supposed to derive from the native enzyme by endogenous proteolysis. The physico-chemical properties of native LeuRS from lactating mammary gland are compared with those of LeuRS's from other sources. 相似文献
19.
The properties of acyl coenzyme A (CoA) synthetase activity were characterized in cultured rabbit coronary microvessel endothelial cells. We report here that microvessel endothelial cells contain two long-chain acyl CoA synthetases. One shows activity with a variety of fatty acids, similar to long-chain non-selective fatty acyl CoA synthetases described previously. The other activity was selective for arachidonic acid and other structurally related substrates. Both activities required ATP, Mg2+ and CoA for optimal activity. The arachidonyl CoA and the non-selective acyl CoA synthetases showed different thermolabilities. Arachidonyl CoA formation was inhibited by greater than 50% after 1 min at 45 degrees C, whereas a 15 min heating treatment was necessary to produce the same relative inhibition of oleoyl CoA synthesis. Glucocorticoid pretreatment (10(-7) M dexamethasone) of the RCME cells did not affect the apparent Km or Vmax, nor the fatty acid selectivity for either acyl CoA synthetase. Therefore, although fatty acyl CoA synthetases may be involved in limiting eicosanoid formation, these activities do not appear to be glucocorticoid-responsive. 相似文献
20.
Molecular weight of T2 NaDNA from viscoelasticity 总被引:1,自引:0,他引:1
The viscoelastic properties of T2 DNA solutions are used to determine the NaDNA molecular weight in four independent ways from the theory of the beads-springs model. The four molecular weights are 131.9, 132.7, 130.5, and 127.6 X 10(6). The average of these values, adjusted for the probable errors in viscoelasticity and concentration measurements, is (126 +/- 5) X 10(6). The four molecular weights are termed Mtaugamma11, Mtaueta, Mtaugamma, and MtauA; each is different in its sensitivity to molecular weight distribution. Their agreement suggests (1) that the theoretical equations relating each M to the corresponding measured properties are valid, (2) that T2 DNA behaves as a partially free-draining polymer chain, and (3) that our solutions were nearly homogeneous in DNA size. We show that serious errors can result if the viscoelastic properties are not extrapolated to their limits at zero shear rate, as well as at zero DNA concentration, before calculating molecular weight. 相似文献