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Role of the major intrinsic protein in lens physiology.

机译：主要内在蛋白在晶状体生理中的作用。

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摘要

The Xenopus oocyte expression system was used to investigate the transport properties of frog lens Major Intrinsic Protein (MIP). SDS-PAGE and immunoblotting demonstrated oocytes injected with MIP mRNA expressed the protein at high levels, and immunolocalization indicated the expressed MIP migrated to the plasma membrane. Both the oocyte water permeability (P{dollar}rmsb{lcub}H2O{rcub}{dollar}) and glycerol permeability (P{dollar}rmsb{lcub}Gly{rcub}{dollar}) were increased in oocytes expressing MIP relative to controls. P{dollar}rmsb{lcub}H2O{rcub}{dollar} was {dollar}3.4times10sp{lcub}-3{rcub} pm0.18times10sp{lcub}-3{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} with MIP vs. {dollar}0.88times10sp{lcub}-3{rcub}pm0.049times10sp{lcub}-1{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} in control oocytes (p {dollar}<{dollar} 0.01). P{dollar}rmsb{lcub}Gly{rcub}{dollar} was {dollar}2.3times10sp{lcub}-6{rcub}pm0.23times10sp{lcub}-6{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} with MIP vs. {dollar}0.92times10sp{lcub}-6{rcub}pm0.086times10sp{lcub}-6{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} in control oocytes (p {dollar}<{dollar} 0.01). These increases in permeability were specific; the oocyte permeability to sucrose, 2-deoxyglucose, inositol, sorbitol, reduced glutathione and urea was unchanged by expression of MIP. The component of P{dollar}rmsb{lcub}Gly{rcub}{dollar} due to MIP was independent of concentration from {dollar}5times10sp{lcub}-5{rcub}{dollar} to {dollar}5times10sp{lcub}-2{rcub}{dollar} M and had a low temperature dependence, suggesting permeation through MIP occurs by diffusion in a water-filled pore, rather than via a carrier mechanism. The P{dollar}rmsb{lcub}Gly{rcub}{dollar} of MIP was inhibited approximately 90% by 1.0 mM Hg{dollar}sp{lcub}++{rcub}{dollar} whereas the P{dollar}rmsb{lcub}H2O{rcub}{dollar} of MIP was not, suggesting water and glycerol have different permeation pathways through MIP.; Expression of MIP enhanced glycerol phosphorylation, the first step in glycerol metabolism, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on a method we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 {dollar}pm{dollar} 0.014 vs. 0.06 {dollar}pm{dollar} 0.004 pmol min{dollar}sp{lcub}-1{rcub}{dollar} cell{dollar}sp{lcub}-1{rcub}{dollar}; p {dollar}<{dollar} 0.01) without changing the binding of glycerol to the kinase (K{dollar}rmsb{lcub}M{rcub}approx12 mu{dollar}M).; Lastly, we performed experiments to determine the significance of these observations to lens physiology. The water permeability of vesicles prepared from rabbit lens was {dollar}5.0times10sp{lcub}-3{rcub}pm2.3times10sp{lcub}-3{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar}. Given the high content of cholesterol, sphingolipid and long-chained, saturated phospholipid in the lens, this value is probably too high to be mediated by the lipid portion of the plasma membrane, suggesting MIP contributes significantly to the water permeability of the lens. Lenses incubated in the presence of {dollar}sp3{dollar}H-glycerol produced {dollar}sp3{dollar}H-glycerol-3-phosphate in the cortex and nucleus, demonstrating glycerol kinase activity in fiber cells. Thus in the lens, MIP may have a role in the processing of glycerol, either for entry into the glycolytic pathway or in the synthesis of lipid.
机译：爪蟾卵母细胞表达系统用于研究青蛙晶状体主要内在蛋白（MIP）的转运特性。 SDS-PAGE和免疫印迹表明，注射MIP mRNA的卵母细胞高水平表达该蛋白，免疫定位表明表达的MIP迁移至质膜。相对于MIP而言，表达MIP的卵母细胞的卵母细胞透水性（P {dollar} rmsb {lcub} H2O {rcub} {dollar}）和甘油渗透性（P {dollar} rmsb {lcub} Gly {rcub} {dollar）均增加。控件。 P {dollar} rmsb {lcub} H2O {rcub} {dollar}为{dollar} 3.4times10sp {lcub} -3 {rcub} pm0.18times10sp {lcub} -3 {rcub} {dollar} cm s {dollar} sp {具有MIP的lcub} -1 {rcub} {dollar}与{dollar} 0.88times10sp {lcub} -3 {rcub} pm0.049times10sp {lcub} -1 {rcub} {dollar} cm s {dollar} sp {lcub}卵母细胞中的-1 {rcub} {dollar}（p {dollar} <{dollar} 0.01）。 P {dollar} rmsb {lcub} Gly {rcub} {dollar}为{dollar} 2.3times10sp {lcub} -6 {rcub} pm0.23times10sp {lcub} -6 {rcub} {dollar} cm s {dollar} sp {具有MIP的lcub} -1 {rcub} {dollar}与{dollar} 0.92times10sp {lcub} -6 {rcub} pm0.086times10sp {lcub} -6 {rcub} {dollar} cm s {dollar} sp {lcub}卵母细胞中的-1 {rcub} {dollar}（p {dollar} <{dollar} 0.01）。这些渗透率的增加是特定的。卵母细胞对蔗糖，2-脱氧葡萄糖，肌醇，山梨糖醇，还原型谷胱甘肽和尿素的通透性通过MIP的表达保持不变。由于MIP，P {dollar} rmsb {lcub} Gly {rcub} {dollar}的成分与浓度从{dollar} 5times10sp {lcub} -5 {rcub} {dollar}到{dollar} 5times10sp {lcub}-无关2 {rcub} {dollar} M且具有较低的温度依赖性，这表明通过MIP的渗透是通过在充满水的孔中扩散而不是通过载体机理发生的。 MIP的P {dollar} rmsb {lcub} Gly {rcub} {dollar}被1.0 mM Hg {dollar} sp {lcub} ++ {rcub} {dollar}抑制约90％，而P {dollar} rmsb { MIP的lcub} H2O {rcub} {美元}并非如此，表明水和甘油通过MIP的渗透途径不同。 MIP的表达增强了甘油磷酸化，这是甘油代谢的第一步，导致甘油掺入脂质的增加。这可能是由于甘油激酶的总活性增加，或者是由于其对甘油的亲和力增加。根据我们目前用来区分这些机制的方法，MIP增加了甘油激酶的最大磷酸化速率（0.12 {pm} pm {dollar} 0.014与0.06 {dollar} pm {dollar} 0.004 pmol min {dollar} sp {lcub } -1 {rcub} {dollar}细胞{dollar} sp {lcub} -1 {rcub} {dollar}； p {dollar} <{dollar} 0.01），而不会改变甘油与激酶的结合力（K {dollar} rmsb {lcub} M {rcub}大约12亩{dollar} M）。最后，我们进行了实验以确定这些观察结果对晶状体生理的重要性。由兔晶状体制备的囊泡的透水度为{dollar} 5.0×10sp {lcub} -3 {rcub} pm2.3×10sp {lcub} -3 {rcub} {dollar} cm s {dollar} sp {lcub} -1 {rcub }{美元}。鉴于晶状体中胆固醇，鞘脂和长链饱和磷脂的含量很高，该值可能太高而无法由质膜的脂质部分介导，这表明MIP显着促进了晶状体的透水性。在{sp3} $ 3H-甘油的存在下孵育的镜片在皮层和细胞核中产生了{sp3} $ 3H-甘油-3-磷酸，证明纤维细胞中的甘油激酶活性。因此，在晶状体中，MIP可能在甘油的加工中起作用，或者进入糖酵解途径，或者在脂质的合成中发挥作用。

著录项

  • 作者

    Kushmerick, Christopher.;

  • 作者单位

    State University of New York at Stony Brook.;

  • 授予单位 State University of New York at Stony Brook.;
  • 学科 Biology Animal Physiology.; Health Sciences Ophthalmology.; Biophysics Medical.
  • 学位 Ph.D.
  • 年度 1996
  • 页码 98 p.
  • 总页数 98
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生理学;生物物理学;
  • 关键词

  • 入库时间 2022-08-17 11:49:22

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