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PubMed2003

The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans.

We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al. [(1999) J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans. Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca(2+), or higher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formation with redox partners were performed in the presence of Ca(2+) in order to simplify the equilibria that had to be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K(d) values of 0.8 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface. For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range. We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase.

Animals↗

PubMed1999

Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution.

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes. This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer. The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0. 32 microM2 for the mutants. Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms. Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A. The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring.

Adenosine Triphosphatases↗

PubMed1999

Self-activation of guanosine triphosphatase activity by oligomerization of the bacterial cell division protein FtsZ.

The essential bacterial cell division protein FtsZ (filamentation temperature-sensitive protein Z) is a distant homologue to the eukaryotic cytoskeletal protein tubulin. We have examined the GTP hydrolytic activity of Escherichia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations greater than 1 microM. At 5 microM FtsZ, we have determined a K(m) of 82 microM GTP and a V(max) of 490 nmol of P(i) min(-1) (mg of protein)(-1). Hydrolysis of GTP requires Mg(2+) and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation experiments show that FtsZ exists as 68% dimer and 13% trimer at 2 microM total protein concentration. Chemical cross-linking of FtsZ also shows that monomer, dimer, trimer, and tetramer species are present at higher (>2 microM) FtsZ concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization. We also monitored the effect of nucleotide and metal ion on the secondary structure of FtsZ; nucleotide yielded no evidence of structural changes in FtsZ, but both Mg(2+) and Ca(2+) had significant effects on secondary structure. Taken together, our results support the hypothesis that Mg(2+)-dependent GTP hydrolysis by FtsZ requires oligomerization of FtsZ. On the basis of these results and structural comparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.

Bacterial Proteins↗

PubMed2004

Thermal and alkaline denaturation of bovine beta-casein.

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.

Animals↗

PubMed1977

Micelles of cerebroside sulphate.

The critical micelle concentration of cerebroside sulphate in water is 0-01 mM: it increases with increasing concentrations of buffer to 0-07 mM in 0-1 M sodium acetate and formate buffers, pH 5-6 and 4-5 respectively. The partial specific volume of the micelles is about 0-94. The behaviour of the micelles in the ultracentrifuge and on Sephadex G-200 shows them to be grossly heterogeneous with respect to size. In 0-1 M buffer s20,w is about 26 S; in water or 0-01 M buffer smaller micelles with an s20,w of about 6 S are also present. In 0-01 M formate, pH 4-5, the smallest species detectable by equilibrium ultracentrifugation had a micellar weight of about 180,000 corresponding to an aggregation number of about 180. Much larger aggregates were also present. It is suggested that the smallest micelles are the substrate for sulphatase A when this is acting as a cerebroside sulphatase in buffers of low ionic strength.

Animals↗

PubMed1978

A new large chylomicron remnant from cholesterol-fed rabbits.

The lipoproteins of density less than 1.063 g/ml of cholesterol-fed rabbits were subjected to analytical ultracentrifugation. In many rabbits two peaks were found in the very low density (Sf greater than 20) portion of the lipoprotein spectrum. They were isolated by preparative ultracentrifugation and analysed. The smaller particles (remnant chylomicrons) had a peak Sf of 37, mean diameter of 36 nm, mean density of 1.00 g/ml, and their chemical composition agreed closely with previous reports. The larger particles had a peak Sf of 270, mean diameter of 80 nm, mean density of 0.97 g/ml and a high (80%) cholesterol ester and low (4%) triglyceride content. The fatty acid composition of the cholesterol esters, phospholipids and triglycerides was similar in both fractions. It is proposed that these large lipoprotein particles are also remnant chylomicrons. Possible reasons are presented to explain the presence of this second peak in the very low density lipoprotein spectrum.

Animals↗

PubMed1989

Antibody to a cryptic, solid phase C1Q antigen in membranoproliferative nephritis.

IgG containing material detected in membranoproliferative nephritis (MPGN) serum with a solid phase (sp) Clq ELISA has been presumed to be immune complexes. However, in serum from 13 MPGN patients containing large amounts of spClq-binding material, sucrose density ultracentrifugation and sieve chromatography showed spClq-binding protein to sediment at 7S or cofractionate with IgG. One serum (stored for 12 years) contained, in addition, spClq-binding material sedimenting at more than 19S. Isolated MPGN IgG was shown to bind to spClq. SpClq-binding material could be totally removed from MPGN serum by absorption with BSA-anti-BSA immune precipitates, and by acid elution of the precipitates IgG binding to spClq could be recovered. F(ab')2, isolated from pepsin digested MPGN IgG, continued to bind spClq. Binding of MPGN IgG or F(ab')2 to spClq was not inhibited by 2 M NaCl. Incubation of MPGN serum with 125I Clq followed by sucrose density ultracentrifugation resulted in a peak of radioactivity at 11S, the sedimentation rate of Clq, giving evidence that material binding fluid phase Clq is not present. SpClq-binding IgG was detected in 54% of 68 MPGN patients. These results indicate that the 7S spClq-binding IgG represents antibody to a cryptic antigen revealed only when Clq fixes to a solid surface.

Antigen-Antibody Complex↗

PubMed2001

A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors.

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50,000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10,000 g for 2 h at 4 degrees C. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3,000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10,000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

Animals↗

PubMed1967

Partial characterization of the sialic acid-free forms of alpha-1-acid glycoprotein from human plasma.

Some of the properties of sialic acid-free alpha(1)-acid glycoprotein prepared by mild acid hydrolysis (pH1.6 at 80 degrees for 1hr.) were compared with those of neuraminidasetreated alpha(1)-acid glycoprotein. Chemically, the former contained less fucose (15%) and amide (2%) residues. Physicochemically, it had undergone certain changes primarily pertaining to the secondary structure, so that the specific optical rotation was more negative than that of the latter. A further expression of this change is probably the difference in the pH range of the resolution into two bands on electrophoresis. The resolution of the glycoprotein prepared by mild acid hydrolysis seems to be extended to more acidic pH values both by starch-gel and free moving-boundary electrophoresis. On ultracentrifugation both preparations appeared homogeneous and sedimented with a rate of 3s. Removal of sialyl residues at different pH values, in the range 1-7, showed that 2moles of sialic acid/mole of protein are very strongly bound. The two variants of alpha(1)-acid glycoprotein were isolated from pooled sialic acid-free alpha(1)-acid glycoprotein by preparative starch-gel electrophoresis and from selected blood of normal adults by fractionation by solubility and chromatography. Ultracentrifugal and starch-gel electrophoretic analyses at pH5, with incubation times of 1 or 24hr., demonstrated that no dissociation-association equilibrium (constant sedimentation coefficient and molecular weight) or isomerization (constant apparent electrophoretic mobilities) exist between the two variants. Therefore these variants are not sub-units of native alpha(1)-acid glycoprotein but represent modifications of naturally occurring proteins. Further, it was shown that the difference in the electrophoretic mobilities between the two variants was not due to any difference in amide content. Immunochemically, the two variants share the same determinants.

Acids↗

PubMed1968

The effect of ribonuclease on rat-liver ribosomes.

1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.

Amino Acids↗

PubMed1968

Purification and properties of molecular-weight variants of human placental alkaline phosphatase.

1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8.6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4.2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.

Albumins↗

PubMed1968

Electron-microscopic and chemical studies of oligomers in horse ferritin.

Horse ferritin was fractionated both by starch-gel electrophoresis and by gel filtration on Sephadex G-200. Monomer fractions contained up to 98% of monomer and oligomer fractions up to 76% of oligomers as determined by quantitative electron microscopy. Percentages obtained from electron micrographs correlated well with analytical starch-gel electrophoretograms and ultracentrifuge patterns. Amino acid analyses of monomer- and oligomer-enriched fractions showed no significant differences. Ferritin oligomers did not apparently dissociate on dilution for electron microscopy or on storage. Apoferritin dimers were stable in 0.01m-phosphate buffer at dilutions down to 0.19mg./ml. as shown by ultracentrifugation. Chemical studies indicated that the intermolecular bonds in oligomers are resistant to a variety of reagents and conditions, including those that would be expected to attack disulphide, peptide and ester linkages respectively. Partial disaggregation was achieved at high pH values and in 67% (v/v) acetic acid. Centre-to-centre intermolecular distances in dimers were found to be about 100å. Three main types of trimer configuration were found and a variety of tetramers and pentamers. These configurations are described and discussed.

Animals↗

PubMed1969

Properties and biosynthesis of a neurotoxic protein of the venoms of s snakes Laticauda laticaudata and Laticauda colubrina.

1. A neurotoxic protein similar to erabutoxins a and b of Laticauda semifasciata was isolated in crystalline form from the venoms of Laticauda laticaudata and Laticauda colubrina. The name ;laticotoxin a' is proposed. 2. Laticotoxin a is homogeneous by CM-cellulose column chromatography, disc electrophoresis and ultracentrifugation and by terminal amino acid analyses. 3. Laticotoxin a consists of 62 amino acid residues. The molecular weight by ultracentrifuging is 6520. 4. The minimal 50% lethal dose of laticotoxin a by intramuscular injection to mice is 0.13mug./g. body wt. The toxin attacks the postsynaptic membrane, competing with acetylcholine. 5. Radioactive amino acids are incorporated into laticotoxin a in vivo. The incorporation is inhibited by puromycin, suggesting that the biosynthesis of the toxin follows the mechanism of protein biosynthesis, although the toxin molecule is rather small as a protein.

Acetylcholine↗

PubMed1973

Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel.

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500-3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800-16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.

Amelogenesis↗

PubMed1974

Characterization of plasma lipoproteins separated and purified by agarose-column chromatography.

1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.

Animals↗

PubMed1978

Comparison of guinea-pig serum lipoproteins after iodination by two different methods.

1. Guinea-pig low-density lipoproteins were isolated by ultracentrifugation and iodinated either by the IC1 method or by the chloramine-T procedure. 2. The efficiency of labelling by both methods was essentially the same. 3. When the two products were compared by ultracentrifugation, gel chromatography and immunodiffusion analysis, no significant difference in their properties was detected. 4. When they were compared by gradient-gel electrophoresis, aggregates were found in the product of the IC1 method, but not in the lipoprotein iodinated by the chloramine-T process. 5. Both products were metabolized by the guinea pig with essentially the same fractional catabolic rate. 6. The fractional catabolic rate of lipoprotein iodinated by the chloramine-T method was not significantly different from that of lipoprotein biologically labelled in the protein moiety with [75Se]selenomethionine. 7. It is concluded that the products of both methods of iodination are almost equally acceptable, provided that the optimum conditions for the chloramine-T reaction are carefully established.

Animals↗

PubMed1979

The role of link-protein in the structure of cartilage proteoglycan aggregates.

Proteoglycan fractions were prepared from pig laryngeal cartilage. The effect of link-protein on the properties of proteoglycan-hyaluronate aggregates was examined by viscometry and analytical ultracentrifugation. Aggregates containing link-protein were more stable than link-free aggregates at neutral pH, at temperatures up to 50 degrees C and in urea (up to 4.0M). Oligosaccharides of hyaluronate were able to displace proteoglycans from link-free aggregates, but not from the link-stabilized aggregates. Both types of aggregate were observed in the ultracentrifuge, but at the concentration investigated (less than 2 mg/ml) the link-free form was partially dissociated and the proportion aggregated varied with the pH and temperature and required more hyaluronate for saturation than did link-stabilized aggregate. The results showed that link-protein greatly strengthened the binding of proteoglycans to hyaluronate and suggest that under physiological conditions it 'locks' proteoglycans on to the hyaluronate chain.

Animals↗

PubMed1980

Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

Animals↗