Insight into nucleotide and Ca²⁺binding to carp ?-actin

Abstract

Nucleotides and Ca 2+ binding to a-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca 2+ was removed with ethylenediaminetetraacetic acid, carp a-actin denatured more rapidly than chicken a-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 ¥ 10 4 /M and 1.2 ¥ 10 5 /M, respectively. Competitive binding of Ca 2+ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraacetic acid (Quin 2) showed that affinity of Ca 2+ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were -65 kJ/mol and -110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.

Thermal stability of carp actin in its polymerized form

“…The difference in thermal stability between these two actins is more pronounced than for G-actins [4]. This is somewhat confusing given that the instability of carp G-actin results from having a low affinity for nucleotides [4,5]. In F-actins, the bound nucleotide cannot be exchanged with free nucleotide in a solution [19].…”

“…The substitution at the 155th position (Ala-155 in carp and Ser-155 in chicken actin) is located in a b-hairpin (Asp154-His161) of the nucleotide binding region, which sandwiches bound nucleotide together with another b-hairpin, Asp11-Lys18 [1]. It has been shown that the Ala-155 is an almost unique substitution that affects the nucleotide binding of carp G-actin [5,21]. Therefore, the open conformation of the nucleotide binding region in carp F-actin and the resulting thermal instability will be due to the substitution.…”

“…Fluorescence labeling of actin with N-(1-pyrene)iodoacetamide was performed according to Kouyama and Mihashi [10] with the modification described previously [5]. Actin bound to 1-N6-ethenoadenosine 5 0 -triphosphate (e-ATP) was prepared as described previously [11].…”

“…The ability to polymerize declines much more rapidly in carp G-actin than in chicken G-actin during heat treatment at between 45 and 55°C [4]. The thermal instability of carp actin is thought to be a result of its low affinity for nucleotides and divalent cations [4,5].…”

Thermal stability of carp G-actin monitored by loss of polymerization activity using an extrinsic fluorescent probe

“…The fact suggests that affinity of ATP for fish actin is lower than that for actin from terrestrial animals. Ooi and Soematsu demonstrated that this is the case for carp α‐actin by directly comparing nucleotide binding between carp and chicken actin 13 . In addition, Ooi and Soematsu showed that affinity of Ca 2+ for carp actin was also lower than that of chicken actin 13 …”