Biomedical subjects
Publications and source records attributed to C Oriol-Audit.
Macrocyclic polyammonium ions induce much more efficient polymerization of actin than the natural polyamines, behaving thus as 'superpolyamines'. The processes observed display large increases in rate, marked structural dependence, macrocyclic and ditopic binding effects and positive cooperativity.
Microinjection of spermine into Xenopus laevis eggs induces precocious furrowing, and together with known spermine-actin interactions, suggests that spermine may be affecting cortical microfilaments involved in cytokinesis. An electron microscopic study of injected eggs revealed that the ultrastructure of the induced furrows was similar to that of both artificially activated eggs and fertilized eggs. In isolated egg cortices, increasing spermine concentrations (1, 3 and 10 mM) resulted in marked changes in cortical microfilament organization. At low concentrations, spermine appeared to stabilize microfilaments and at higher concentrations induced lateral associations between filaments and formation of bundles. The actin nature of these cortical microfilaments was confirmed by immunocytochemistry. The electrophoretic profiles of proteins from control and spermine-treated isolated cortices were similar. Although the total protein content of isolates in 3 and 10 mM spermine was elevated, the relative actin content remained constant. The results are in agreement with previous in vitro studies of polyamine interactions with actin and support the hypothesis that a polyamine-actin interaction may be important in the regulation of cytokinesis.
The involvement of polyamines in cytokinesis has been examined in eggs of the amphibian X. laevis. Microinjection of spermine or spermidine into unfertilized eggs induced a shortening of the first division cycle and early formation of cleavage-like constrictions. Eggs were activated by injection and developed furrows about 45 min later, whereas the first division normally occurred around 120 min after activation. In terms of concentration, spermine was slightly more effective than spermidine, but putrescine had no influence on the division cycle.
Electron microscopy of negatively stained samples shows that spermine and spermidine induce the polymerization of G-actin into filaments and paracrystalline bundles. In the presence of low concentrations of polyamines (0.02 mM spermine or 0.2 mM spermidine), filaments resembling salt-induced F-actin were observed, but as the concentration of polyamine was increased, relatively unordered bundles appeared. At about 0.2 mM spermine or 2.5 mM spermidine, the width of the bundles increased and they appeared more ordered with paracrystalline regions. This change was correlated with the gelation of the actin-polyamine mixture. At higher concentrations of polyamines (greater than 5 mM spermine or greater than 8 mM spermidine), the bundles had a similar ordered structure but, instead of gelation, there was an immediate precipitation of actin bundles. Spermine and spermidine also promote bundle formation from salt-induced F-actin. Two major paracrystalline forms were observed. Type I resembles the Hanson-type paracrystals induced by magnesium. Type II, characterized by an axial striation of 5.9 nm appears to be unique to polyamine-induced actin bundles.
Explore the source record for details and available documents.
Addition of KCl to a solution of synthetic myosin minifilaments in 10 mM citrate-Tris buffer (pH 8.0) induces the growth of filaments. These filaments, at pH 8.0, resemble in their morphological and hydrodynamic properties the synthetic filaments described by Josephs and Harrington [Josephs, R., & Harrington. W. F. (1966) Biochemistry 5, 3474--3487]. The rate of filament growth depends critically on the KCl concentration in the solution. Low rates of filament formation are noted in the presence of both low (below 80 mM KCl) and high (above 0.15 M KCl) salt concentrations, whereas at the intermediate KCl concentrations the filaments are formed at a fast rate. The formation of filaments from minifilaments is a reversible process, and under moderate salt concentrations, these two polymeric systems appear to exist in a dynamic equilibrium. Small amounts of minifilaments can induce rapid polymerization of dissociated myosin; i.e., they can act as a seeding material. These and other observations are discussed in terms of a direct route for filament formation from myosin minifilaments.
Morphologically similar short myosin and rod filaments (minifilaments) have been prepared in 10 mM Tris--citrate buffer, pH 8.0, in the absence of other myosin or rod forms. Both minifilament systems are dissociated in the same manner in the presence of ATP or pyrophosphate. Identical binding of these ligands to myosin and rod minifilaments suggests that myosin heads play no role in substrate-induced destabilization of the minifilaments. The effects of ATP and pyrophosphate on minifilaments are similar to their dissociating effect on synthetic filaments [Harrington, W. F., & Himmelfarb, S. (1972) Biochemistry 11, 2945--2952], thus justifying their use in conformational studies in lieu of filaments. In view of their small size and homogeneity, the minifilaments constitute an appropriate material for such studies. The binding of pyrophosphate to myosin and rod minifilaments decreases their alpha-helical content, as measured by circular dichroism. No change in the secondary structure of subfragment 1 and light meromyosin is observed upon binding of pyrophosphate, but substantial changes (10%) are detected in subfragment 2. The structural changes in myosin, possibly relevant to contraction, are localized in the subfragment 2 region of the molecule. These results emphasize the importance of charge interactions in the functional behavior of thick filaments.
The interaction of alkali light chains of vertebrate skeletal myosin with nucleotides and KCl has been examined by chemical modifications of these proteins, by direct binding measurements, and in circular dichroism studies. The reactivities of the single thiol groups in the isolated alkali light chains A1 and A2 have been studied by reacting these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). MgATP and MgADP reduced the reactivities of thiol groups while high concentrations of KCl increased them. Subsequent equilibrium dialysis experiments verified the presence of a low-affinity nucleotide binding site per each alkali subunit. Circular dichroism measurements revealed that KCl induced local (around phenylalanines) and overall (alpha-helical content) conformational changes of equal magnitude in the two alkali light chains. However, salt induced different conformational changes in the subfragment 1 isoenzymes, S-1(A1) and S-1(A2). This differential salt effect on the S-1 isoenzymes was confirmed by comparing their thermal stability in different salt conditions. AT low KCl concentrations (5 mM), S-1(A1) was found to be considerably more heat labile than S-1(A2); at higher salt levels (50 mM KCl) the stability of S-1(A1) approached that of S-1(A2). These experiments are discussed in terms of the relationship between the alkali subunits and the ATP and the actin-binding sites of myosin.
Experiments have been carried out to assess the involvement of the myosin light chains [obtained by treatment of myosin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2)] in the control of cross-bridge movement and actomyosin interactions. Chymotryptic digestions of myosin, actomyosin, and myofibrils do not detect any Ca2+-induced change in the subfragment 2 region of myosin. Actin, like Ca2+, protects the in situ Nbs2 light chains from proteolysis and causes a partial switch in the digestion product of myosin from subfragment 1 to heavy meromyosin. This effect is independent of the state of aggregation of myosin, and it persists in acto heavy meromyosin and in actinomyosin in 0.6 M NaCl. Digestions and sedimentation studies indicate that there is no direct acto light chain interaction. Proteolysis of myosin shows a gradual transition from production of heavy meromyosin to subfragment 1 with lowering of the salt level. In the presence of Ca2+ heavy meromyosin is generated both in digestions of polymeric and of monomeric myosin. These results are explained in terms of localized changes within the Nbs2 light chains and subfragment 1. Subunit interactions in the myosin head lead to a Ca2+-induced reduction in the affinity of heavy meromyosin for actin in the presence of MgATP. The resulting Ca2+ inhibition of the actin-activated ATPase of myosin can be detected at high salt concentrations(75 mM KCl).
Explore the source record for details and available documents.
Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain length of the polyamine. Among the biological polyamines tested, spermidine and spermine are the most efficient. The polymerization of actin can also be induced by the corresponding mono or diguanidine derivatives of these polyamines but monoamines or amino acids are inactive at the same concentration. The transformation of actin from a globular to a fibrous from upon addition of spermidine is also demonstrated by the changes in the near-ultraviolet circular dichoroic spectrum of this protein. Moreover, the polyamine-induced F -actin exhibits the same properties as the salt-induced F -actin: it strongly activates the Mg2+ -ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.
Explore the source record for details and available documents.