Silva A Madhushika M, Goonasekara Charitha L, Hayley Michael, Heeley David H
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada.
Department of Biochemistry, Faculty of Medicine, Kotelawala University, Colombo 10390, Sri Lanka.
Biochemistry. 2020 Dec 22;59(50):4725-4734. doi: 10.1021/acs.biochem.0c00882. Epub 2020 Dec 8.
The phosphorylated and unphosphorylated forms of tropomyosin Tpm1.1(α) are prepared from adult rabbit heart and compared biochemically. Electrophoresis confirms the high level of enrichment of the chromatography fractions and is consistent with a single site of phosphorylation. Covalently bound phosphate groups at position 283 of Tpm1.1(α) increase the rate of digestion at Leu-169, suggestive of a conformational rearrangement that extends to the midregion. Such a rearrangement, which is supported by ellipticity measurements between 25 and 42 °C, is consistent with a phosphorylation-mediated tightening of the interaction between various myofilament components. In a nonradioactive, co-sedimentation assay [30 mM KCl, 1 mM Mg(II), and 4 °C], phosphorylated Tpm1.1(α) displays a higher affinity for F-actin compared to that of the unphosphorylated control (, 0.16 μM vs 0.26 μM). Phosphorylation decreases the concentration of thin filaments (pCa 4 plus ATP) required to attain a half-maximal rate of release of product from a pre-power stroke complex [myosin-S1-2-deoxy-3--(-methylanthraniloyl)ADP-P], as investigated by double-mixing stopped-flow fluorescence, suggestive of a change in the proportion of active (turned on) and inactive (turned off) conformers, but similar maximum rates of product release are observed with either type of reconstituted thin filament. Phosphorylated thin filaments (pCa 4 and 8) display a higher affinity for myosin-S1(ADP) versus the control scenario without affecting isotherm steepness. Specific activities of ATP and Tpm1.1(α) are determined during an incubation of rat cardiac tissue [12 day-old, 50% phosphorylated Tpm1.1(α)] with [P]orthophosphate. The incorporation of an isotope into tropomyosin lags behind that of ATP by a factor of approximately 10, indicating that transfer is a comparatively slow process.
从成年兔心脏中制备原肌球蛋白Tpm1.1(α)的磷酸化和非磷酸化形式，并进行生化比较。电泳证实了色谱级分的高度富集，且与单个磷酸化位点一致。Tpm1.1(α)第283位的共价结合磷酸基团增加了Leu-169处的消化速率，这表明构象重排延伸至中间区域。这种重排在25至42°C之间的椭圆率测量中得到支持，与磷酸化介导的各种肌丝成分之间相互作用的收紧一致。在非放射性共沉降试验[30 mM KCl、1 mM Mg(II)和4°C]中，与未磷酸化对照相比，磷酸化的Tpm1.1(α)对F-肌动蛋白显示出更高的亲和力（分别为0.16 μM和0.26 μM）。通过双混合停流荧光研究发现，磷酸化降低了从预动力冲程复合物[肌球蛋白-S1-2-脱氧-3-(-甲基蒽酰胺基)ADP-P]中获得产物半最大释放速率所需的细肌丝浓度（pCa 4加ATP），这表明活性（开启）和非活性（关闭）构象体比例发生了变化，但两种类型的重组细肌丝观察到的产物最大释放速率相似。磷酸化的细肌丝（pCa 4和8）对肌球蛋白-S1(ADP)的亲和力高于对照情况，且不影响等温线斜率。在大鼠心脏组织[12日龄，50%磷酸化的Tpm1.1(α)]与[P]正磷酸盐孵育期间，测定了ATP和Tpm1.1(α)的比活性。同位素掺入原肌球蛋白的过程比掺入ATP的过程滞后约10倍，这表明转移是一个相对缓慢的过程。
