Roth D A, Brooks G A
Department of Physical Education, University of California, Berkeley 94720.
Arch Biochem Biophys. 1990 Jun;279(2):377-85. doi: 10.1016/0003-9861(90)90505-s.
To study the kinetics of lactate transport in an isolated, nonmetabolizing system, skeletal muscle sarcolemmal membrane vesicles were purified from 22 female Sprague-Dawley rats. L(+)-[U-14C] Lactate at 10 concentrations demonstrated saturation kinetics with a Vmax of 139.4 nmol/mg/min, and an apparent Km of 40.1 mM. Threefold higher initial rates of L(+)-lactate uptake were seen at 37 degrees C than at 25 degrees C, indicating temperature sensitivity. Transport was stereospecific for the L(+) isomer: isotopic D(-) uptake rates remained linear at concentrations from 1 to 200 mM, and 1 mM D(-) remained 6-fold lower in net uptake after 60 min than the L(+) isomer. Furthermore, unlabeled 10 mM D(-)-lactate in the external medium could only inhibit 1 mM isotopic (L(+) uptake by 12%, whereas unlabeled 10 mM L(+)-lactate and pyruvate inhibited 82 and 71%, respectively. Additionally, 10 mM beta-hydroxybutyrate and acetoacetate could moderately inhibit (27 and 32%, respectively) 1 mM L(+)-lactate transport, but the unsubstituted aliphatic monocarboxylates (formate, acetate, propionate), tricarboxylic acid cycle intermediates (malate, succinate, oxaloacetate, alpha-ketoglutyrate, citrate), amino acids (alanine, aspartate, glutamate), and palmitate or adenosine in 10-fold excess could not effectively inhibit 1 mM L(+)lactate uptake under cis-transport conditions. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid could inhibit L(+)-lactate transport by only 13%, so that lactate transport does not appear to be affected directly by Cl- or HCO3- fluxes. It was demonstrated that KCl could not evoke a membrane potential-induced overshoot of lactate uptake in the presence or absence of valinomycin. Moreover, gluconate could substitute for Cl-, indicating that Cl- flux does not contribute to a membrane potential-dependent component of the transport mechanism, suggesting an electroneutral translocation process. Protein-modifying reagents significantly inhibited 1 mM L(+)-lactate transport during pH-stimulated conditions (p-chloromercuriphenyl-sulfonic acid, 83%; N-ethylmaleimide, 86%; HgCl2, 56%; mersalyl, 63% inhibition). We conclude that the skeletal muscle lactate transporter is a membrane-bound protein, specifically associated with the sarcolemma, that demonstrates saturation kinetics, competition, stereospecificity, and sensitivity to temperature as well as various ionic cis-inhibitors. The lactate transporter is a potentially important regulator of lactate flux across skeletal muscle, and may help to regulate intracellular pH and intermediary metabolism during lactic acidosis.
为了研究在一个分离的、非代谢系统中乳酸转运的动力学，从22只雌性斯普拉格-道利大鼠中纯化出骨骼肌肌膜囊泡。10种浓度的L(+)-[U-14C]乳酸表现出饱和动力学，Vmax为139.4 nmol/mg/分钟，表观Km为40.1 mM。在37℃时L(+)-乳酸摄取的初始速率比25℃时高3倍，表明其对温度敏感。转运对L(+)异构体具有立体特异性：在1至200 mM的浓度下，同位素D(-)摄取速率保持线性，并且在60分钟后，1 mM D(-)的净摄取量比L(+)异构体低6倍。此外，外部介质中未标记的10 mM D(-)-乳酸仅能抑制1 mM同位素（L(+)摄取的12%，而未标记的10 mM L(+)-乳酸和丙酮酸分别抑制82%和71%。另外，10 mMβ-羟基丁酸和乙酰乙酸可适度抑制（分别为27%和32%）1 mM L(+)-乳酸转运，但未取代的脂肪族单羧酸盐（甲酸、乙酸、丙酸）、三羧酸循环中间产物（苹果酸、琥珀酸、草酰乙酸、α-酮戊二酸、柠檬酸）、氨基酸（丙氨酸、天冬氨酸、谷氨酸）以及10倍过量的棕榈酸或腺苷在顺式-转运条件下不能有效抑制1 mM L(+)乳酸摄取。4,4'-二异硫氰基芪-2,2'-二磺酸仅能抑制L(+)-乳酸转运的13%，因此乳酸转运似乎不受Cl-或HCO3-通量的直接影响。已证明在有或没有缬氨霉素的情况下，KCl都不能引起膜电位诱导的乳酸摄取过冲。此外，葡萄糖酸盐可替代Cl-，表明Cl-通量对转运机制中膜电位依赖性成分没有贡献，提示存在电中性转运过程。蛋白质修饰试剂在pH刺激条件下显著抑制1 mM L(+)-乳酸转运（对氯汞苯磺酸，83%；N-乙基马来酰亚胺，86%；HgCl2，56%；汞撒利，63%抑制）。我们得出结论，骨骼肌乳酸转运体是一种膜结合蛋白，与肌膜特异性相关，表现出饱和动力学、竞争性、立体特异性、对温度以及各种离子顺式抑制剂的敏感性。乳酸转运体是骨骼肌中乳酸通量的潜在重要调节因子，可能有助于在乳酸酸中毒期间调节细胞内pH和中间代谢。
