Microtubule protein polymerization

Consideration of membrane as a target for chemotherapeutic drugs has been reviewed and relevant studies with cisplatin summarized [79]. The amino acid uptake mechanism in LI 210 cells is affected by cisplatin [80] and platinum complexes inhibit plasma membrane phosphatase activity in ascites cells [81]. Microtubule protein polymerization is also affected adversely [82]. Effects on mitochondrial functions and properties have been examined [83—86], along with studies on inhibition of sulfhydryl-containing enzymes [87—90]. 

As noted above, turbidity and polymer weight concentration can be directly related (Cp = or, where t is the turbidity), and the proportionality constant may be determined experimentally (cf. Zackroff et al., 1980). Microtubule protein preparations, however, usually contain a fraction of protein that does not contribute to polymer formation, and the most likely interpretation is that this fraction is composed of assembly-incompetent tubulin and nontubulin protein contaminants (Gaskin et al., 1974 Zackroff et al., 1980). Note that Eq. (30) is based on the assumption that Co represents active, polymerization-competent protomer. If only a fraction y, less than one, is active, this equation must be corrected to give... 

Free protein monomers of intermediate filaments rarely occur in the cytoplasm, in contrast to microfilaments and microtubules. Their polymerization leads to stable polymers that have no polarity. 

The threshold concentration of monomer that must be exceeded for any observable polymer formation in a self-assembling system. In the context of Oosawa s condensation-equilibrium model for protein polymerization, the cooperativity of nucleation and the intrinsic thermodynamic instability of nuclei contribute to the sudden onset of polymer formation as the monomer concentration reaches and exceeds the critical concentration. Condensation-equilibrium processes that exhibit critical concentration behavior in vitro include F-actin formation from G-actin, microtubule self-assembly from tubulin, and fibril formation from amyloid P protein. Critical concentration behavior will also occur in indefinite isodesmic polymerization reactions that involve a stable template. One example is the elongation of microtubules from centrosomes, basal bodies, or axonemes. 

The generality of the end-wise depolymerization kinetic model is indicated by the comparison of the observed and predicted time-courses of cold-induced microtubule disassembly (Fig. 3). See Self-Assembly Protein Polymerization... 

Any polymerization reaction in which the product of each elongation step can itself also undergo further polymerization. When the same types of bonds and/or conformational states that are present in the reactant(s) are generated within product(s) during elongation, the process is referred to as isodesmic polymerization. Such is the case for the indefinite polymerization of actin, tubulin, hemoglobin S, and tobacco mosaic virus coat protein. See Nudeation Protein Polymerization Actin Assembly Kinetics Microtubule Assembly Kinetics Microtubule Assembly Kinetics... 

Turbidity has proven to be especially useful in studies of protein polymerization, where one can demonstrate that the turbidity is directly proportional to the polymer mass concentration. This is illustrated in the following plot (Fig. 1) obtained for assembled microtubules. 

PROTEIN POLYMERIZATION ACTIN ASSEMBLY KINETICS MICROTUBULE ASSEMBLY KINETICS... 

ACTIN ASSEMBLY KINETICS MICROTUBULE ASSEMBLY KINETICS PROTEIN POLYMERIZATION KINETICS NUCLEIC ACID RENATURATION KINETICS Nucleic acid structure,... 

Induction of polymerization of porcine brain microtubule protein by 5 pM of test compound relative to the effect of 25 pM of Epo B, which gave maximal polymerization (85% of protein input). 

Factors influencing the polymerization of outer fibre microtubule protein. 

Microtubules are polymeric fibers that play a key role in cell division and in cell shape and motility. They are formed from subunits of the protein tubulin (molecular weight about 100 kd), which assemble into protofilaments. Each microtubule is composed of thirteen linked protofilaments. Intermediate in size are oligomers, which are short stretches of protofilaments (Correia and Williams, 1983). The assembly of microtubules is driven by the hydrolysis of the nucleotide guanosine triphosphate (GTP), which binds to the tubulin subunits and hydrolyzes to the diphosphate (GDP) on polymerization. The GDP can also bind to tubulin, but it inhibits microtubule assembly. The nucleotides are rapidly exchangeable when bound to tubulin, but become nonexchangeable when attached either to oligomers or to microtubules. 

Certain proteins endow cells with unique capabilities for movement. Cell division, muscle contraction, and cell motility represent some of the ways in which cells execute motion. The contractile and motile proteins underlying these motions share a common property they are filamentous or polymerize to form filaments. Examples include actin and myosin, the filamentous proteins forming the contractile systems of cells, and tubulin, the major component of microtubules (the filaments involved in the mitotic spindle of cell division as well as in flagella and cilia). Another class of proteins involved in movement includes dynein and kinesin, so-called motor proteins that drive the movement of vesicles, granules, and organelles along microtubules serving as established cytoskeletal tracks. ... 

Centrosomes, also called the microtubule organizing centre, are protein complexes that contain two cen-trioles (ringlike structures) and y- tubulin. They serve as nucleation points for microtubular polymerization and constrain the lattice structure of a microtubule to 13 protofilaments. They are responsible for organizing the mitotic spindle during mitosis. 

Vinca alkaloids are derived from the Madagascar periwinkle plant, Catharanthus roseus. The main alkaloids are vincristine, vinblastine and vindesine. Vinca alkaloids are cell-cycle-specific agents and block cells in mitosis. This cellular activity is due to their ability to bind specifically to tubulin and to block the ability of the protein to polymerize into microtubules. This prevents spindle formation in mitosing cells and causes arrest at metaphase. Vinca alkaloids also inhibit other cellular activities that involve microtubules, such as leukocyte phagocytosis and chemotaxis as well as axonal transport in neurons. Side effects of the vinca alkaloids such as their neurotoxicity may be due to disruption of these functions. 

Microtubule-associated proteins bind to microtubules in vivo and subserve a number of functions including the promotion of microtubule assembly and bundling, chemomechanical force generation, and the attachment of microtubules to transport vesicles and organelles (Olmsted, 1986). Tubulin purified from brain tissue by repeated polymerization-depolymerization contains up to 20% MAPs. The latter can be dissociated from tubulin by ion-exchange chromatography. The MAPs from brain can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 

Just as myosins are able to move along microfilaments, there are motor proteins that move along microtubules. Microtubules, like microfilaments, are polar polymeric assemblies, but unlike actin-myosin interactions, microtubule-based motors exist that move along microtubules in either direction. A constant traffic of vesicles and organelles is visible in cultured cells especially using time-lapse photography. The larger part of this movement takes place on micrombules and is stimulated by phorbol ester (an activator of protein kinase C), and over-expression of N-J aj oncoprotein (Alexandrova et al., 1993). 

---