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Molecular weight dependency of heparin inhibition of microtubule assembly in vitro
Abstract:
Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.
Insights
Heparin inhibits microtubule assembly by binding to microtubule-associated proteins, preventing proper structure formation. Taxol can reverse this inhibition, though resulting microtubules show altered protein content.
Area of Science:
- Biochemistry
- Cell Biology
Background:
- Microtubules are essential cytoskeletal components involved in cell structure and division.
- Microtubule assembly is regulated by microtubule-associated proteins (MAPs).
Purpose of the Study:
- To investigate the effect of heparin on microtubule assembly in vitro.
- To elucidate the mechanism by which heparin influences microtubule formation and stability.
Main Methods:
- In vitro assembly assays using bovine brain microtubule proteins.
- Analysis of heparin's interaction with microtubule-associated proteins and tubulin.
- Assessment of heparin's effect on critical concentration and microtubule structure.
Main Results:
- Low concentrations of heparin inhibited microtubule assembly and disassembled preformed microtubules.
- Microtubule-associated proteins counteracted heparin's inhibitory effects.
- Heparin's inhibition was dependent on its concentration and molecular weight, not total protein concentration.
- Taxol reversed heparin's inhibition, but resulted in aberrant microtubules with reduced MAPs and incorporated heparin.
Conclusions:
- Heparin inhibits microtubule assembly primarily by binding to MAPs, disrupting their function.
- The heparin-MAP complex cannot support microtubule nucleation or stabilization.
- Heparin's molecular weight influences its inhibitory potency.
- Taxol can overcome heparin-induced inhibition, but alters microtubule composition.