[10] Light scattering and differential refractometry

Pittz, Eugene P.; Lee, James C.; Bablouzian, B.; Townend, Robert; Timasheff, Serge N.

doi:10.1016/s0076-6879(73)27012-x

Cited by 37 publications

(19 citation statements)

References 60 publications

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“…When proteins are noninteracting, the scattering becomes directly proportional to the concentration of proteins and their Stokes radii. However, a deviation from linearity with increasing concentra-tions of the protein provides an indication of association-dissociation (32,33). Scattering was measured in a Hitachi F-4500 spectrofluorometer at 25°C.…”

Section: Methodsmentioning

confidence: 99%

A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

Chakraborty¹,

Das²,

Datta³

et al. 2002

Journal of Biological Chemistry

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Disaggregation and reactivation of aggregated proteins by chaperones is well established. However, little is known regarding such kind of function of single-domain small cyclophilins (CyPs). Here we demonstrate that, with increasing concentrations, fully active adenosine kinase (AdK) of Leishmania donovani tends to form soluble aggregates, resulting in inactivation. Using this inactive enzyme as the substrate, it is shown that a CyP from L. donovani (LdCyP) alone can cause complete disaggregation, leading to reactivation of the enzyme. The reactivating ability of LdCyP remains unaffected even in the presence of cyclosporin A and macromolecular crowding agents. The reactivation occurs noncatalytically and is reversible. A truncated LdCyP, devoid of 88 amino acids from the N terminus, is found to be required in near stoichiometric proportion to reactivate AdK, suggesting essentiality of the C-terminal region. Gel filtration and light-scattering experiments together with protein cross-linking studies revealed that both full-length LdCyP and the truncated form directly interact with AdK and convert oligomeric forms of the enzyme to monomeric state. Homology modeling studies suggest that the exposed hydrophobic residues of LdCyP, by interacting with solvent-accessible hydrophobic surface of AdK, pull apart its aggregated inactive oligomers to functional monomers. Clearly, the results are consistent with the interpretation that the higher efficiency of the truncated LdCyP is most likely due to increased exposure of the hydrophobic residues on its surface. These observations, besides establishing L. donovani AdK as one of the model enzymes to study aggregation-disaggregation of proteins, raise the possibility that single-domain small CyPs, under physiological conditions, may regulate the activity of aggregation-prone proteins by ensuring their disaggregation.

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Section: Methodsmentioning

confidence: 99%

A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

Chakraborty¹,

Das²,

Datta³

et al. 2002

Journal of Biological Chemistry

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“…The presence of inactive tubulin would also increase the apparent critical concentration: no change is seen between CM 7 and CMR79, and the critical concentration is decreased in CMR795. We therefore deduce that the scattering intensity is reduced, possibly due to changes in hydration or ion association with tubulin (29).…”

mentioning

confidence: 86%

Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

Keates¹,

Sarangi²,

Ling³

1981

Proc. Natl. Acad. Sci. U.S.A.

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We have examined mutant lines of Chinese hamster ovary cells that have increased resistance to the antimicrotubule drug Colcemid. Analysis of the functional properties of purified microtubule protein indicates that increased tolerance to the drug in vivo is reflected in altered properties of microtubules and tubulin in vitro. In this study, we have examined one series of related mutants and have found different microtubule alterations associated with each selection step. These changes include decreased Colcemid-binding affinity, an altered electrophoretic pattern of tubulin subcomponents, increased resistance to Colcemid inhibition of polymerization in vitro and, in one case, a de-.creased critical concentration for microtubule assembly. Characterized mutants of the class described here will be useful for probing the regulation of microtubule assembly in vivo.Microtubules are a major component ofthe mitotic spindle and cytoskeleton of eukaryotic cells (1). A characteristic of microtubules is their ability to assemble and disassemble under different conditions. Considerable effort has been expended on elucidating the control mechanisms for microtubule assembly (1-3). Microtubules are formed by polymerization of the globular protein, tubulin, and appear to be regulated by association with a variety ofadditional proteins, the microtubule-associated proteins (MAPs) (2). Several workers have developed kinetic models to describe the microtubule elongation reaction in vitro (4, 5), but in vivo regulation of assembly, in particular with respect to initiation, is less well understood (3). We feel that a genetic approach may provide an effective probe of regulatory mechanisms in vivo.To this end, we have isolated mutant clones of Chinese hamster ovary (CHO) cells resistant to Colcemid, with the expectation that these cells would possess altered microtubules (6). We report here the characterization of the properties of microtubules isolated from a related series of Colcemid-resistant mutants. The selection procedures used were designed to avoid the membrane mutants that have reduced drug permeability previously isolated (7,8 Microtubule protein was isolated from cells grown in monolayer culture, which were washed twice in phosphate-buffered saline and three times in an isotonic buffer of 0.2 M 2-(N-morpholino) ethanesulfonic acid (MES), pH 6.6/1 mM MgCl2/1 mM ethylene glycol bis(,/3aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/2 mM 2-mercaptoethanol. To the washed cell pellet was added 0.7 vol of 1 mM MgCl2/L mM EGTA/2 mM 2-mercaptoethanol/1.7 mM GTP/0. 1 mM L-1-tosylamido-2-phenylethyl chloromethyl ketone/0.1 mM N-tosyl-L-lysine chloromethyl ketone adjusted to pH 6.6 with NaOH. Cells were broken by ultrasonic cavitation for three 1-min bursts at 0C (MSE sonicator). The suspension was centrifuged at 81,000 X gma for 40 min at 0C. Glycerol was added to the supernatant as recommended by Nagle et al. (9) to a final concentration of 5 M, and'GTP was added to a final concentration of 2.7 mM. The mixture was incub...

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“…The occurrence of IAA binding was manifested by a sharp increase in light scattering. This may indicate that, upon IAA binding, significant changes took place in some physical parameters involved in the process of light scattering, such as the polarizability, the refractive index, or the radius of gyration, that are closely linked to the overall structural property of a protein (22). The results of CD experiments also appeared to conform to the concept of IAAinduced conformational alteration.…”

Section: Iaa Binding Induces a Conformational Change Of Abp 57 -mentioning

confidence: 72%

A Soluble Auxin-binding Protein, ABP57

Kim

Min

Kim

et al. 2001

Journal of Biological Chemistry

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ABP 57 is an auxin-binding protein that possesses receptor function. In this study, a protocol for ABP 57 purification was developed on the basis of cross-reactivity shown between ABP 57 and antisera raised against bovine serum albumin, which enabled us to purify ABP 57 with a high yield and to further characterize it. ABP 57 activates plant plasma membrane H ؉ -ATPase (PM H ؉ -ATPase) via direct interaction. The binding of indole-3-acetic acid (IAA) to the primary binding site on ABP 57 caused a marked increase in the affinity of ABP 57 for PM H ؉ -ATPase, which was accompanied by a change in ABP 57 conformation. Meanwhile, additional IAA binding to the secondary site on ABP 57 nullified the initial effect without inducing further conformational change. When ABP 57 with IAA occupying only the primary site interacted with PM H ؉ -ATPase, no IAA could access the secondary site. These results suggest that IAA-induced biphasic alteration in the affinity of ABP 57 for PM H ؉ -ATPase correlates with a bell-shaped dose response of the enzyme to IAA. There is also a possibility that, whereas the stimulation phase of the response is associated with a conformational change of ABP 57 , the destimulation phase probably results from hindrance arising directly from the presence of IAA at the secondary site. Auxin-binding proteins (ABPs)1 are a class of low abundance proteins in plants that bind active auxins with high affinity and specificity. As a result of ABP-auxin binding, ABP might initiate the auxin signal pathways leading to various cellular responses and thus have a plant hormone receptor function. Extensive studies have led to the identification of a number of ABPs in both membrane and soluble fractions, of which the best characterized in terms of cellular location, biochemical nature, and putative receptor function is ABP1 (for reviews, see Refs. 1-5). The main natural auxin in most plants is indole-3-acetic acid (IAA), which appears to exert many effects in plants, including, most importantly, cell elongation. In fact, the term "auxin" is used to describe chemical substances that stimulate elongation growth in coleoptiles and many stems.There is no clear explanation of the mechanism by which auxin regulates cell growth. The immediate effect of exposure of plant tissues to auxin is proton excretion, occurring within minutes. The resulting apoplastic acidification provides a favorable condition for cell wall loosening, which could be an early part of auxin-induced cell expansion (1, 3, 6). H ϩ -ATPase activity of the plasma membrane (PM) in plants is responsible for proton extrusion from the cell. Therefore, the acidification of the cell wall space is thought to result from activation of the electrogenic H ϩ -ATPase in some manner. There is evidence that auxin stimulates plant PM H ϩ -ATPase, which is correlated with an increased affinity of the enzyme for ATP (7) but does not involve a direct effect of auxin on the enzyme itself (8, 9).We have previously isolated two isoforms of a soluble ABP in rice plants, one fr...

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[10] Light scattering and differential refractometry

Cited by 37 publications

References 60 publications

A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

A Single-domain Cyclophilin from Leishmania donovaniReactivates Soluble Aggregates of Adenosine Kinase by Isomerase-independent Chaperone Function

Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

A Soluble Auxin-binding Protein, ABP57

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