Change In Sedimentation Coefficient Research Articles

Observation of Contraction and Expansion in a Bis(peptide)‐Based Mechanical Molecular Actuator

A novel, bis(peptide) based molecular actuator (1) has been synthesized. It is demonstrated to undergo contraction and expansion controlled by the addition and removal of Cu2+; this is demonstrated by the direct observation of a change in hydrodynamic properties by using sedimentation analysis and size exclusion chromatography. The molecule undergoes a large change in sedimentation coefficient, axial ratio, and size exclusion chromatography elution time when it binds copper. The demonstration of a controlled change in the mechanical properties of 1 make it a good starting point for the development of molecular devices that will harness changes in molecular shape and size to create molecular devices such as sensors or valves.

Regulated Conformation of Myosin V

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.

Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.

Thermodynamic nonideality as a probe of allosteric mechanisms: preexistence of the isomerization equilibrium for rabbit muscle pyruvate kinase.

Sedimentation velocity studies in the presence and absence of an inert space-filling solute, sucrose, have been used to establish preexistence of the isomerization equilibrium responsible for the allosteric behavior of rabbit muscle pyruvate kinase. Whereas the inclusion of phenylalanine (5 mM) with enzyme gives rise to a decrease of 0.3 S in the sedimentation coefficient of pyruvate kinase, the corresponding effect of phosphoenolpyruvate is to increase the sedimentation coefficient by 0.03 S. Consideration of these findings to signify the existence of an isomeric equilibrium between compact and expanded forms of the enzyme is substantiated by the finding that inclusion of sucrose (0.1 M) also brings about the change in sedimentation coefficient effected by phosphoenolpyruvate. By demonstrating that rabbit muscle pyruvate kinase undergoes isomerization in the absence of substrate, this study removes any necessity to consider the existence of an isomerization equilibrium that is substrate-induced; and thereby provides experimental support for adoption of the Monod model of allostery to interpret enzyme kinetic data for pyruvate kinase [R. W. Oberfelder, B. G. Barisas, and J. C. Lee (1984) Biochemistry 23, 3822-3826].

Chromatin structure. Evidence that the 30-nm fiber is a helical coil with 12 nucleosomes/turn.

Sedimentation analysis has been used to compare the structure of 30-nm chromatin fibers, isolated and digested under conditions that maintain the native structure, with relaxed-refolded chromatin. The native chromatin fibers show sharp, ionic strength-dependent changes in sedimentation coefficient that are not apparent in relaxed-refolded fibers. The first transition at approximately 20 mM ionic strength reflects the organization of the 10-nm polynucleosome chain into a loose helically coiled 30-nm fiber. Between 20 and 60 mM ionic strength there is considerable interaction between nucleosomes within the coils to generate a stable helical array with 12 nucleosomes/turn. Above 60 mM ionic strength the helical coil continues to condense until it precipitates at ionic strengths slightly greater than those considered physiological, indicating that there is no end point in fiber formation. The data is incompatible with a solenoid model with 6 nucleosomes/turn and also rules out the existence of a beaded subunit structure.

A conformational transition in gizzard heavy meromyosin involving the head-tail junction, resulting in changes in sedimentation coefficient, ATPase activity, and orientation of heads.

Gizzard heavy meromyosin (HMM) sediments in the ultracentrifuge as a single peak, whose sedimentation coefficient (S20,w) decreases from 9 to 7.5 S upon increasing the NaCl concentration from 0.02 to 0.3 M. This decrease is accompanied by a parallel increase in Mg2+-ATPase activity, suggesting that both changes have a common molecular basis. Phosphorylation decreases S20,w and increases ATPase activity, while ATP increases S20,w. Sedimentation equilibrium studies indicate that HMM undergoes no detectable aggregation at 0.02 or 0.4 M NaCl, remaining monomeric with a molecular weight of 3.4 X 10(5). In contrast, S20,w of subfragment 1 does not change with changes in ionic strength, and its ATPase activity does not decrease at low ionic strengths. Electron micrographs of samples of HMM prepared at low ionic strength show that up to half of the molecules are flexed, i.e. the heads are bent at the neck and project back toward the tail, while the remaining molecules have either one or both of the heads pointing away from the tail. In samples prepared at high ionic strength only about 10% of the molecules are flexed. There is a linear relationship between the fraction of flexed molecules and S20,w, with no significant bending or folding of the tail and no detectable change in the shape of the heads. This correlation suggests that the changes in ATPase activity and S20,w may be a result of the reorientation of the heads.

The androgen receptor in normal human foreskin

Abstract Use of glycerol and the protease inhibitor phenylmethylsulfonyl fluoride resulted in a fourfold increase in the yield of androgen receptor from human neonatal foreskin and a tenfold increase in stability of this receptor. The general physical properties of the androgen-binding component isolated from the cytosol of human neonatal foreskin were found to be consistent with those previously reported for androgen receptors. The synthetic androgen methyltrienolone (R1881) was bound with high affinity (a mean dissociation constant of 0.51 nmol/L) and low capacity (5.6 fmol/mg of protein); maximum binding required 4 hours. Affinity of the receptor was highest for R1881 followed by dihydrotestosterone, testosterone, and progesterone. 17β-Estradiol showed little or no competition. The receptor exhibited a change of sedimentation coefficient from 8S to 4S upon treatment with 0.1 mol/L potassium chloride. Isoelectric focusing demonstrated the existence of two molecular species with apparent isoelectric points of 7.2 and 5.7.

Physico-chemical properties of the hydroxysterol binding protein of human lymphocyte cytosol: Effects of high salt concentrations and molybdate

Side chain-hydroxylated derivatives of cholesterol (OH sterol) inhibiting lymphoblastic transformation bind with high affinity and specificity to a hydroxysterol binding protein (OHSBP) in the cytosol of human lymphocytes. These binding properties of OHSBP suggested some analogies with that of steroid hormone receptors. The observation of a nuclear binding of 25-OH[ 3H]cholesterol prompted us to apply to the cytosolic OH sterol-OHSBP complex the physico-chemical treatments known to ‘activate’ the steroid hormone receptors. A change of sedimentation coefficient from 8.3 to 4.3 S was observed in hypertonic buffer (0.4 M KC1) but the resulting 4.3 S complex dissociates easily whereas the ‘native’ 8.3 S form does not. Moreover, molybdate did not prevent the 8.3→ 4.3 S transformation induced by KCland neither ammonium sulfate precipitation nor increasing temperature had any effect on the sedimentation coefficient of the 8.3 S complex. Thus, several physico-chemical features differentiate the OH sterol-OHSBP complex from steroid hormone receptors. Hydroxysterol Human lymphocyte Specific binding protein Cell division

Nuclear [ 3H]4-hydroxytamoxifen (4-OHTAM)- and [ 3H]estradiol (E 2)-estrogen receptor complexes in the MCF-7 breast cancer and GH 3 pituitary tumor cell lines

Nuclear [ 3H]4-OHTAM-ER complexes extracted by 0.6 M KC1 from the MCF-7 human breast cancer and the GH 3 rat pituitary tumor cell lines, sedimented as a 5S form on sucrose gradients after 1 to 24 h exposure to ligand (20 nM). The nuclear [ 3H]4-OHTAM-ER from MCF-7 cells increased from 2 + 0.2 pmoles/mg DNA at l h to approximately 4 + 0.1 pmoles/mg DNA at 6 and 24 h. In the GH 3 cells the nuclear binding of [ 3H]4-OHTAM increased rapidly, and there was no significant difference in the receptor levels at 1 and 6 h (10 ± 1.3 and 8.9 ± 1.1 pmoles/mg DNA, respectively). At 24 h there was a decrease in [ 3H]4-OHTAM-ER levels (7.0 ± 0.2 pmoles/mg DNA); however, this was due primarily to an increase in DNA synthesis, which occurred in these cells by 24 h. It appears that in both cell lines there is no processing of nuclear [ 3H]4-OHTAM-ER, and it is not associated with changes in sedimentation coefficient of the complex. When both cell lines were incubated with[ 3H]E 2 (20 nM), processing of the nuclear [ 3H]E 2-ER occurred over 6 h. In the MCF-7 cells there was a decrease in receptor content within 6 h from 3.2 ± 0.2 to 0.9 ± 0.2 pmoles/mg DNA. The nuclear [ 3H]E 2-ER in the GH 3 cells decreased from 7.3 ± 0.5 to 2.8 + 0.3 pmoles/mg DNA. Although these cell lines appeared to process the [ 3H]E 2-ER complex, the nuclear forms of [ 3H]E 2-ER were different. In the GH 3 cells the nuclear [ 3H]E 2-ER consistently sedimented as a 5S complex whereas the nuclear receptor from MCF-7 cells sedimented mainly as a 4S complex. Occasionally (20% incidence) both 4S and 5S [ 3H]E 2-ER forms were observed which were recognized by a monoclonal antibody (D547) raised to the ER. These data suggest that the activity of estradiol and 4-OHTAM in vitro may be more related to nuclear processing, or lack of it, than to the physical form of the ligand-receptor complex.

Intestinal 1, 25-dihydroxyvitamin D 3 receptors: Hormone-dependent uptake and saturability of nuclear components in vitro

An in vitro system has been designed to study the interaction between intestinal cytoplasmic 1, 25-dihydroxyvitamin D receptors and highly purified homologous nuclei. Cytosolic receptors, labeled with 1, 25-dihydroxyvitamin D, specifically associate with intestinal nuclei as ternary chromatin complexes, an event which proceeds in the absence of a demonstrable change in sedimentation coefficient. This interaction is a saturable process with a dissociation constant of 2.2 × 10 −10 M at 0° C, and is specific for the 3.3S cytoplasmic receptor since neither the 4.1S nor the 6S vitamin D binding proteins associate in a similar fashion. In addition, the interaction is hormone-dependent, since free receptors also do not bind. The development and characterization of an in vitro system such as this is the beginning in an attempt to assess the molecular action of these receptors on intestinal nuclei.

Dissociation of chitosomes by digitonin into 16 S subunits with chitin synthetase activity

Digitonin exerts profound effects on chitosomes (microvesicular structures with chitin synthetase activity isolated from the fungus Mucor rouxii). At low concentrations, it stimulates chitin synthetase (UDP-2-acetamido-2-deoxy- D-glucose : chitin 4-β-acetamidodeoxy- D-glucosyltransferase, EC 2.4.1.16) activity; at higher concentrations, it inhibits it. Digitonin also causes disintegration of the chitosome and the release of a homogeneous population of chitosome subunits with chitin synthetase activity. These chitosome subunits have a sedimentation coefficient of 16 S, compared to 105 S for whole chitosomes, as determined by centrifugation in sucrose density gradients, and measure 7–12 nm in diameter. After dissociation, chitin synthetase remains in a zymogenic state, and requires treatment with a protease for activation. No change in sedimentation coefficient of chitosome subunits was observed after proteolytic activation. The product synthesized by the chitosome subunits was characterized by X-ray diffractometry as α-chitin and was by this criterion indistinguishable from chitin made by preparations of undissociated chitosomes. However, in the electron microscope, the chitin microfibrils made from chitosome subunits were, in general, much shorter than those produced by undissociated chitosomes and often exhibited a needle-like appearance.

Some Effects of pH, Salt, Urea, Ethanediol and Sodium Dodecyl Sulphate on Tobacco Necrosis Virus

SUMMARY The sedimentation coefficients of tobacco necrosis virus strain D (TNV d ) in sodium phosphate at pH 5 and pH 7 are 104S and 97S respectively. The change in sedimentation coefficient between pH 5 and 7 is fully reversible and the decrease on transfer to pH 7 is unaffected by the presence of Mg2+. TNV d is disrupted by Bacillus subtilis protease at pH 7.5 but not at pH 5; ribonuclease neither disrupts virions nor destroys the infectivity of TNV d at either pH 7.5 or pH 5. Virions are markedly unstable at alkaline pH but the effectiveness with which different buffers dissociate TNV d varies. Virus is largely resistant to dissociation by 1 m-NaCl, 1 m-urea, 10 m-ethanediol and 1% sodium dodecyl sulphate (SDS) at or below pH 6 and is largely susceptible to dissociation by these agents at or above pH 7. Our results suggest that as the pH is lowered some non-covalent bonds important for virion stability may be formed and/or some electronic repellent interactions causing virion instability may be weakened.

An explanation for rotor speed effects observed during sedimentation of large folded DNA molecules

A model is proposed to account for the changes in sedimentation coefficient ( s) that are observed when isolated bacterial micleoids are sedimented at different rotor speeds. It is suggested that the terminal of DNA loops in the chromosome become analogous hydrodynamically to the ends of a linear DNA double-helix. The loops then become more extended at higher rotor speeds resulting in an increased fractional coefficient. Application of the Zimm (1974) equation to this model shows that the predicted changes in s values are consistent with those observed. Similar changes in s values occur when the chromosome is sedimented in rotors of different geometry including a zonal rotor. Thus, the changes are not due to wall interactions.

Difference sedimentation velocity adapted to low molecular weight proteins

The difference sedimentation velocity technique reported by Kirschner and Schachman (1971, Biochemistry 10, 1900–1919) has been modified to eliminate the need for a supernatant region. The method is now applicable to the measurement of small changes in sedimentation coefficient for low molecular weight proteins and other small macromolecules. Procedural changes necessary to overcome the absence of a supernatant region until late in the run have been devised and tested. A modified double-sector centerpiece was used to match the radial positions of the two menisci. The integration of the moment of the concentration difference was carried out from the meniscus to the plateau region, rather than over the peak only. The interference baseline was measured on photographs at the start of each run and after remixing. Some instability of baseline height was noted. The calculation method adjusted the baseline height to correspond with the concentration difference in the plateau region arising from unequal radial dilution. Tests of the method have been made using D 2O to retard the sedimentation of lysozyme. The interference results at low D 2O concentration (small values of Δs) are in agreement with schlieren results at high D 2O concentrations. Changes of 0.005 S have been detected.

Cytosol and nuclear estrogen receptor in the genital tract of the rhesus monkey

Estrogen receptor levels of cytosol and nuclei were measured in the cervix, vagina, myometrium, and endometrium of the rhesus monkey after the following experimental treatments: (1) castration; (2) castration with estradiol treatment; and (3) castration with estradiol plus progesterone treatment. Plasma hormone levels similar to those of normal menstrual cycles were achieved by implantation of silastic capsules containing estradiol or progesterone. The K D, 4 × 10 −9 M, for the cytosol receptor was similar in all tissues. Competition studies showed cytosol receptor affinities to be E 2 > e 1 > E 3 > DES. Cytosol receptor levels in estrogenized animals were lowered by progesterone treatment. Progesterone treatment did not alter the levels of nuclear receptor indicating that the lower levels of cytosol receptor obtained with progesterone were not due to nuclear translocation of cytosol receptor. Estrogen treatment changed the sedimentation coefficient of the endomentrial and myometrial cytosol receptor from 4 s to 8 s when compared with the castrate. This alteration in sedimentation coefficient was blocked by progesterone treatment. Changes in sedimentation coefficient were not observed in the cervix or vagina. Nuclear receptor in estrogen and estrogen plus progesterone treated animals sedimented as a 5 s protein. The data suggest that both qualitative and quantitative changes in the estradiol cytosol receptor of the genital tract are induced by progesterone and that these alterations may be related to the hormone interactions in target organs.

Allosterism, regulation and cooperativity: The case of ribonucleotide reductase of Lactobacilus leichmannii

The binding of modifiers and substrates to the ribonucleotide reductase from Lactobacillus leichmannii has been studied with the use of enzyme highly purified by a modified preparative procedure. This monomeric enzyme represents a simpler system than classical allosteric enzymes since the study of allosterism, regulation, and conformational responses in the latter is complicated by interaction between subunits. Analysis of earlier kinetic results indicates that ribonucleotide substrates bind weakly to the regulatory site (K D for GTP, 4 m m) compared with binding to the catalytic site (K D for GTP 0.24 m m), and in so doing produce substrate activation. The apparent rate constant for product formation when GTP is not bound to the regulatory site is 31% of that observed when GTP occupies the regulatory site. Part of this effect is due to tighter binding of coenzyme when substrate is bound to the regulatory site, a result confirmed by direct measurement of adenosylcobalamin binding. The effect of modifiers such as dGTP which accelerate the reduction of specific substrates is also in part due to increased coenzyme binding as measured both kinetically and by equilibrium dialysis and ultrafiltration methods. The modifiers also seem to have an obligatory role in the enzyme mechanism. Without their presence neither formation of the radical-pair intermediate from adenosylcobalamin nor associated reactions (coenzyme degradation, coenzyme H-exchange with water) occur under the experimental conditions observed. When modifier concentration dependence for these reactions is compared with estimates of equilibrium constants for dissociation of modifiers and substrates from complexes with the regulatory and catalytic sites it appears likely that binding at the regulatory site is responsible. Calculation of coenzyme saturation under the experimental conditions indicates, however, that insignificant amounts of coenzyme are bound to the enzyme except when the regulatory site is occupied, and this effect adequately explains the necessity for modifiers. Binding of dGTP does not alter the circular dichroism of the enzyme in the far-ultraviolet region (200 to 250 nm) but causes significant changes in the aromatic region, and therefore appears to perturb tryptophyl and tyrosyl residues though not necessarily directly. Measurements have been made of the fractional change in sedimentation coefficient due to dGTP binding to the enzymes by the use of an adjustable Raleigh mask and double sector cell in the Beckman model E ultracentrifuge. Values for Δs/ s of −1.14% at 22° and −1.16% at 37° were obtained. The former, after correction for the fractional increase due to the calculated effect of dGTP on the buoyant weight of the protein, gives a value of (Δs/ s corr = −2.84% . If this is due to increase in hydrodynamic volume of the protein alone, it corresponds to an 8.8% increase. If it were entirely due to increased departure from spherical shape it would correspond to a change in axial ratio from 2.9 to 3.2. In the presence of saturating concentrations of ATP and dihydrolipoate and the maximum permissible concentration of a coenzyme analogue Δs/ s was −0.33%, the corrected value being about −1.99% at 37°. These conformational responses are as great as determined for multimeric enzymes such as in the effect of CTP on aspartate transcarbamylase, and are consistent with relay of the regulatory signal over a considerable intersite distance.

Assembly of a spherical plant virus.

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 degrees C and did not require the presence of Mg2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.

Stepwise Degradation of Serum Low Density Lipoprotein by Sodium Dodecyl Sulfate1

The structure of human serum low density lipoprotein (LDL) was investigated by perturbing the LDL structure with sodium dodecyl sulfate (SDS). The change in LDL structure induced by the addition of SDS was monitored by sedimentation velocity measurements, ultraviolet difference spectroscopy, fluorescence spectroscopy and proteolytic digestion of apo-LDL with subtilisin BPN' [EC 3.4.21.14]. As the concentration of SDS was increased from 0.1 mg/ml to 3 mg/ml with LDL concentrations between 2.0 mg/ml and 4.4 mg/ml, the sedimentation coefficient of LDL changed in three distinct steps. It was found by chemical analyses that not more than 30% of the total lipid was lost from LDL in the second step, whereas the final step in the change of sedimentation coefficient corresponded to the complete removal of apo-LDL from the constituent lipids of LDL. The ultraviolet difference spectrum between the native and SDS-treated LDL and the quenching of LDL fluorescence underwent about 80% of the total change while the SDS concentration was only sufficient to cause the second of the three step changes in sedimentation coefficient. SDS-polyacrylamide gel electrophoresis of apo-LDL treated with subtilisin BPN' also showed that more than 70% of apo-LDL became susceptible to proteolysis under the same conditions. These results were interpreted as indicating that the solubilization of 20 to 30% of the lipids on the surface of LDL exposed nearly 80% or more of apo-LDL to the solvent. A small portion of apo-LDL was, however, still firmly anchored to the remaining lipid micelle as long as the concentration of SDS was less than that required to cause the final step of the change in sedimentation coefficient.

Sedimentation of DNA from Human Fibroblasts Irradiated with Ultraviolet Light: Possible Detection of Excision Breaks in Normal and Repair-Deficient Xeroderma Pigmentosum Cells

Usual alkaline sucrose techniques, which produce single-stranded DNA from unirradiated mammalian cells of $2-5\times 10^{8}$ daltons (120-165 S) prove inadequate for resolution of strand breaks transiently present during excision repair of ultraviolet (uv) damage. These breaks seem to be relatively few in number and sedimentation changes are at the limit of resolution. Using a simple modification employing brief lysis in alkali, DNA with sedimentation coefficients up to 350 S have been obtained from human fibroblasts. 350-S DNA from unirradiated fibroblasts consists of complementary strands cosedimenting with choline-labeled material originating in the cytoplasm, in a conformation that allows renaturation to double-stranded DNA on neutralization after gradient fractionation. Large changes in sedimentation coefficient occur immediately after irradiation of normal fibroblasts with doses as low as $5-10 {\rm ergs}/{\rm mm}^{2}$ , but not xeroderma pigmentosum (XP) fi...

Sequential conformational changes with the binding of NAD + to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The technique of differential sedimentation has been used to examine the titration of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in D 2O buffer with NAD +. The fractional change in sedimentation coefficient was shown to increase linearly as a function of ligand saturation. An examination of the concentration dependence of the sedimentation coefficients of the apo- and holo-enzymes excluded the possibility of changes in the state of aggregation of the enzyme with ligand binding. It was then possible to interpret the changes in sedimentation coefficient with ligand binding in terms of changes in frictional coefficient, reflecting a contraction in the tetrameric assembly on binding NAD +. The linear relation observed between the degree of ligand saturation and the degree of change in frictional coefficient was consistent with the sequential mechanism of conformational changes.