关于光合作用光系统Ⅱ(PS-Ⅱ)电子传递的研究

在菠菜叶绿体和光系统Ⅱ颗粒中,DCIP和铁氰化钾的光还原对DCMU或Tris洗涤的抑制作用反应不同,其影响取决于pH(Tris)和浓度(DCMU)。在Tris的pH为8.0时,叶绿体和光系统Ⅱ颗粒的DCIP光还原全部被Tris(0.8M)洗涤抑制,而仍保留有少量残留的铁氰化钾光还原活力。在正常的叶绿体中,DCMU在5×10~(-5)M和5×10~(-4)M的浓度下,DCIP的光还原活力全部丧失,而铁氰化钾的光还原活力分别保留11.5％和10.8％。用Tris洗过的叶绿体,当DCIP的光还原活力被5×10~(-6)M,5×10~(-5)M和5×10~(-4)M的DCMU全部抑制时,铁氰化钾的光还原活力分别保留有对照的14.l％,15.0％和13.5％。在正常的光系统Ⅱ颗粒中,DCIP的光还原全部被5×10~(-6)M和5×10~(-5)M的DCMU抑制,而分别保留有13.8％和11.7％残留的铁氰化钾的光还原活力。用Tris(pH 7.6,0.8M)洗涤过的光系统Ⅱ颗粒,在5×10~(-7)M和5×10~(-6)M DCMU浓度下,残留的铁氰化钾光还原活力是26.2％和19.2％,而DCIP的光还原全部被抑制。 Tris洗涤过的或DCMU处理过的叶绿体和光系统Ⅱ颗粒残留的铁氰化钾光还原活力在短波光(651毫微米)下比在长波光(714毫微米)下高。讨论了光系统Ⅱ中可能包含有两个短波光反应。     

乙酸苯汞对棉花气孔开放、蒸騰作用和光合作用的影响

1.以1×10~(-4)、3.3×10~(-4)和10×10~(-4)M三种浓度的乙酸苯汞(PMA)分别喷洒在棉叶上,1小时后即可使气孔接近完全关闭,这种影响3、5日后消失。 2.用PMA处理的叶片,蒸腾作用显著降低。浓度越高,影响越大;而作用的时间也越长。10×10~(-4)MPMA处理的叶子,19天以后还比对照部位的蒸腾作用降低约10％。 3.3.3×10~(-4)M PMA对光合作用的影响不太明显。浓度为10×10~(-4)M时,初期的影响也不大;但3日后使光合作用降低1/3,叶缘素的含量也下降;甚至处理后的第10天,处理部位的光合作用强度仍然显著地比对照低。 4.对PMA作用的性质、使用的适宜浓度以及存在的问题提出了初步的讨论。     

麻黄碱对兔主动脉和心房作用机制的研究

麻黄碱(E,×10~(-4)—3.3×10~(-3)M)和去甲肾上腺素(NA,6×10~(-8)—6×10~(-5)M)均能引起离体兔主动脉条浓度依赖性收缩。可卡因能明显地增强 NA 的作用,但明显地减弱麻黄碱的作用,用可卡因后麻黄碱的作用为用可卡因前的10—92.6%。利血平处理后,麻黄碱和 NA的作用都明显增强,但利血平对前者的增强作用比后者更甚。在利血平处理及未处理肌条上,麻黄碱的作用均可被酚妥拉明阻断。麻黄碱(3.3×10~(-6)—3.3×10~(-5)M)和异丙肾上腺素(ISP,10~(-9)—10~(-5)M)均能引起离体兔心房率的增加。可卡因明显地减弱麻黄碱的这一作用,即为对照组的8.8—29.1%,但不影响 ISP 的作用。利血平处理后,麻黄碱对兔心房的作用也明显减弱,为对照纽的15.4—28.4%;而 ISP 作用则略增加。3×10~(-4)麻黄碱可明显增加[3~H]NA 从兔主动脉条的流出量,此作用在给药后5min内即开始,可持续30 min 以上。上述结果提示,对于兔主动脉和心房,麻黄碱兼具直接作用于效应器细胞和通过释放末梢中 NA 的间接作用;直接作用在主动脉占优势,间接作用在心房占优势。     

麻黄碱对小鼠输精管平滑肌的作用

麻黄碱(2×10~(-4)-1.6×10~(-3)M)和去甲肾上腺素(6×10~(-7)-7.5×10~(-5)M)均能引起并增强离体小鼠输精管自发性收缩。酚妥拉明(10~(-6)M)可明显对抗麻黄碱的作用。小鼠经利血平处理后,输精管对去甲肾上腺素的反应有所增强,但不明显影响麻黄碱的作用。在半钙营养液(含CaCl_21.25mM)中,麻黄碱的作用明显减弱;在无钙溶液中作用完全消失。但去甲肾上腺素的作用在半钙营养液中不明显减弱;无钙溶液中仍有大部分标本对去甲肾上腺素产生微弱反应。钙道阻断剂戊脉安可明显减弱或取消麻黄碱兴奋小鼠输精管的作用,而对抗去甲肾上腺素的作用较弱。麻黃碱在较低浓度(5×10~(-7)-1.6×10~(-5)M)时,尚能抑制输精管对电场刺激所致收缩反应,这种抑制作用可被α_2受体对抗剂育亨宾(10~(-7)M)所对抗。降低营养液中钙离子浓度,可增强麻黄碱的抑制作用。高浓度麻黄碱完全抑制电场刺激所致收缩,而出现明显增强的自发收缩。以上表明,麻黄碱对小鼠输精管突触前膜α_2受体和突触后膜α_1受体都有激动作用。麻黄碱引起并增强输精管平滑肌收缩的作用,主要是其对突触后α_1真受体的直接作用,并需要有钙离子的参与。     

离体绿豆胚轴在吸胀过程中蛋白质合成与ATP水平及能荷值的关系

绿豆胚轴吸胀过程中 ATP 水平及能荷(E.C.)值的上升伴随着蛋白质合成速率的增加。用5×10~(-5)M 和5×10~(-4)DNP 处理后,组织中 ATP 水平及能荷值降低,同时也抑制了~3H-亮氨酸参入 TCA 不溶性蛋白质的量。并观察到 CCCP(1×10~(-3)M 和1×10~(-4)M)有类似作用。以0.2μg/ml 亚胺环己酮处理,蛋白质合成下降69%,ATP 和 E.C.都略有增加;用1μg/ml 和5μg/ml 处理,蛋白质合成接近停止(占总合成量的6—7%),ATP 水平稍有下降,E.C.值仍保持不变。用每毫升含1μg 和10μg 放线菌素 D 处理,使蛋白质合成分别降低23%和48%,而 ATP 水平及 E.C.值不受任何影响。这些结果表明绿豆胚轴吸胀初期,其蛋白质合成受 ATP 水平及能荷值的调节,而腺苷酸库和能荷变化都不受放线菌素 D 的影响。     

重组人红细胞生成素单克隆抗体的制备

本文应用常规淋巴细胞杂交瘤技术制备了4株能稳定分泌抗人重组红细胞生成素(rHuEPO)单克隆抗体(McAb)的小鼠杂交瘤细胞系:BⅡ1B5、DⅡ6B9、MⅡ1H4、GⅠ3E7,用鼠单克隆抗体分型试剂盒鉴定,其分泌的McAb的类型分别是:IgM、IgM、IgG1和IgG2a。间接ELISA法测定细胞上清的效价为1×10~(-2)～1.25×10~(-1),腹水效价为1×10~(?)～1×10~(?)。培养上清经ELISA鉴定,与IL-2、GM-CSF、IFN-α等细胞因子均无交叉反应。只与rHuEPO特异性结合。     

家蝇卵巢中保幼激素结合蛋白的研究(英语)

本文运用活性炭结合试验测定了家蝇卵巢中保幼激素结合蛋白(JHBP)的含量.卵巢中JHBP对JHⅢ有较高的结合活性,其结合常数为2.1×10~(-8)M,~3H保幼激素Ⅲ(~3H-JH)和JHBP的结合可被未标记的JH Ⅲ抑制,但保幼激素的类似物ZR 512或ZR515均无抑制效应.卵巢中JHBP的含量在家蝇羽化后48小时达最大值,是羽化后60小时、72小时家蝇卵巢中JHBP含量的6.5倍和15.5倍.羽化后24小时、36小时的家蝇卵巢无JHBP的结合活性.若刚羽化的家蝇用JHⅢ处理,36小时后,JHBP则可检测到.本实验的结果表明卵巢中JHBP浓度的变动可能和JH对家蝇卵黄发生的作用有关.     

磁共振弥散加权成像及ADC值对鼻咽癌颅底放疗疗效的评价价值

目的:探讨磁共振弥散加权成像(DWI)及表观扩散系数(ADC)值在鼻咽癌颅底放疗中的临床价值。方法:收集我院于2013年6月~2014年6月复查的40例鼻咽癌患者,分别于放疗前及放疗结束12个月以后对所有患者行常规核磁共振成像(MRI)及DWI检查,测量放疗前、后ADC值,根据影像学检查以及临床诊断结果分为复发组(n=5)及未复发组(n=35)。结果:复发组放疗前ADC值为(0.797±0.031)×10~(-3)mm~2/s,与未复发组放疗前ADC值(0.805±0.028)×10~(-3)mm~2/s比较,差异无统计学意义(P0.05)。复发组放疗结束12个月以后ADC值为(1.097±0.091)×10~(-3)mm~2/s,与未复发组放疗结束12个月以后ADC值(1.705±0.128)×10~(-3)mm~2/s比较,差异有统计学意义(P0.05)。结论:DWI作为一种新兴的磁共振成像技术,对于鼻咽癌颅底放疗疗效的评价具有重要价值,通过DWI对ADC值的测量,可有效的预测患者预后是否良好。     

雌性高氏后异刺线虫在生殖周期不同阶段的细胞核内Feulgen-DNA含量的变化

(1)应用细胞光度法测定表明,雌性高氏后异刺线虫在第一次生殖周期中,卵原细胞核DNA含量从5.47×10~(-12)克(即3.77C)下降到1.12×10~(-12)克(即0.77C)。作为对照的雄虫并无生殖周期,其精原细胞DNA含量稳定于6.18×10~(-12)克(即4.26C)。这与Ⅰ期雌虫的卵原细胞接近。(2)雌虫在第一次生殖轮回的前三期中,受精囊内精子的DNA含量为1.49×10~(-12)克(即1.03G)。进入Ⅳ期后,精子缩小,Feulgen反应减弱。直到第二次生殖轮回时,受精囊的精子也只有0.36×10~(-12)克(即0.25C)。雄虫储精囊的精子含DNA 1.38×10~(-12)克(即0.96C),这与前三期雌虫受精囊的精子相等。(3)Ⅳ期雌虫子宫内幼虫细胞的Feulgen染色有深浅之分,分别含DNA1.29×10~(-12)克(0.89C)和0.64×10~(-12)克(0.44C)。当排完幼虫后,雌虫卵巢又可排出少数卵,并受精发育。产生的胚胎也有深浅两种细胞,分别含1.35×10~(-12)克(0.93C)和0.67×10~(-12)克(0.39C)DNA。上述核内。DNA含量的变化是否的确反映细胞基因组大小的改变,还需进一步分析DNA顺序复杂性来证实。(4)前三期雌虫中,含有大量DNA的肠细胞不再渗入氚标记胸苷,而Ⅳ期雌虫的双倍体肠细胞能小量渗入。至于卵原细胞和精原细胞则经常合成DNA。     

顺铂与小鼠艾氏腹水肝癌细胞膜的相互作用

本文研究了顺铂对小鼠艾氏腹水肝癌细胞膜蛋白内源性荧光的淬灭作用和测定了其在膜上的结合量。结果表明顺铂能与癌细胞膜结合。按存在两类结合部位,得到表观结合常数和结合部位数为: K_1=1.35×10~5L/mol n_1=6.80×10~(-4)mol/g(protein) K_2=2.50×10~3L/mol n_2=1.92×10~(-3)mol/g(protein)     

Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.     

Some biological properties of human chorionic follicle stimulating hormone.

The biological properties of human chorionic FSH (hCFSH) for rat ovaries were investigated. Highly purified hCFSH had similar response to the ovarian augmentation test as bovine FSH and significantly enhanced 3H-thymidine uptake by granulosa cells and theca cells in the ovary of hypophysectomized rat. In contrast, highly purified hCG little responded to the ovarian augmentation test and had no effect on 3H-thymidine uptake by the ovary. These results indicate that hCFSH may promote the follicular growth of ovary resulting from granulosa cell proliferation and its enlargement. In addition, freshly harvested porcine granulosa cells were employed in an in vitro system to investigate specific binding of hCFSH to ovarian receptor. Radioiodinated hCFSH (125I-hCFSH) and hCG (125I-hCG) were respectively incubated with cell suspensions. Binding of these hormone preparations was proportional to the cell number and increased with the time of incubation through 120 minutes. The binding ability of 125I-hCFSH to the cells was greater than that of 125I-hCG. Increasing concentrations of unlabeled hCFSH in the incubation mixture progressively inhibited the uptake of 125I-hCFSH by granulosa cells. Unlabeled hCG was not able to compete with 125I-HCFSH binding. The similar phenomenon to inhibit the binding of 125I-hCG to the cells was also recognized in the presence of unlabeled hCG. These findings suggest that granulosa cell has at least two different types of receptor sites: one for hCFSH and the other for hCG.     

Luteinizing Hormone Receptors are Confined in Mesoscale Plasma Membrane Microdomains Throughout Recovery from Receptor Desensitization

We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80–160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.     

Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum

The level of hCG/LH receptor has been shown to undergo marked changes during the life span of rat corpus luteum. To evaluate whether these fluctuations are due to changes in the receptor subunit structure or receptor protein content, the 125I-hCG binding activity and the receptor subunit structure were determined during different time periods of pseudopregnancy. The maximum 125I-hCG binding activity was observed on day 7, after which it decreased by 20 and 45% on day 11 and day 14, respectively. The Scatchard analysis of 125I-hCG binding data showed that the decrease in binding activity was caused by a change in the number of binding sites rather than a change in the binding affinity. The LH/hCG receptor in ovarian membranes obtained on days 7, 11 and 14 were then characterized by the method of affinity cross-linking. All four subunits of the LH/hCG receptor were detected in the ovarian membranes at all stages while the intensity decreased parallel to a decrease in hCG binding from day 7 to day 14. These results suggest that the decrease in 125I-hCG binding activity in rat ovarian membranes from day 7 to day 14 of pseudopregnancy is due to a decrease in receptor concentration rather than a change in the receptor subunit structure.     

The Effects of Neuraminidase and Gangliosides on Ovarian LH/hCG Receptors During Rat Development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6–8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (K_D) of about 10⁻⁹ M. The K_D of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10⁻¹⁰ M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptors in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

Epidermal growth factor receptors in fetal and maternal rabbit lung

The pattern of morphologic and functional development of lung during intrauterine period is influenced by several endogenous compounds. Recently Epidermal Growth Factor (EGF), when administered in vivo, has been shown to accelerate pulmonary maturation in fetal rabbit and sheep. We sought evidence for EGF receptor occurrence in fetal and maternal rabbit lung plasma membranes. The percent specific binding (mean ± S.E.M.) (125-I) EGF to LPM in the mother (n=5) and the fetus at term (n=7) was 1.08 ± 0.08 and 2.25 ± 0.12 per 175 μg of LPM protein respectively. The number of receptor sites per mg of LPM protein in the mother were significantly less than that in the fetus (44 ± 11 and 250 ± 24 × 10^?10, p < 0.001) with no apparent differences in Kd (2.10 ± 0.39 and 2.47 ± 0.24 × 10⁹). Presence of high affinity receptors for EGF in fetal and maternal lung plasma membranes suggests a direct role of EGF in fetal lung maturation.     

The effects of neuraminidase and gangliosides on ovarian LH/hCG receptors during rat development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6-8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (KD) of about 10(-9) M. The KD of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10(-10) M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptor in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops)

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for ¹²⁵I-hCG, (0.9–2.5 × 10⁹ M^?1, and 0.7–1.64 × 10⁹ M^?1 respectively, for green and rhesus monkeys) and low binding capacity (0.343–0.682 fmol/mg and 0.198–0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 10⁹ M^?1) and low binding capacity (1.04 fmol/mg tissue homogenate) for ¹²⁵I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.     

Covalent Cross-Linking of Radiolabeled Human Chorionic Gonadotropin to Rat Ovarian Luteinizing Hormone Receptor with Glutaraldehyde

Abstract

Crude plasma membranes of pseudopregnant rat ovaries were incubated with ¹²⁵I-labeled human chorionic gonadotropin (¹²⁵I-hCG) and the formed luteinizing hormone (LH)/hCG receptor-¹²⁵I-hCG complex was solubilized, partially purified by Sepharose 6B gel filtration, cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. An apparent molecular weight (mol wt) of 130,000 was obtained for the non-reduced complex. A similar-sized complex was observed, when ³H-hCG (β-subunit labeled) instead of ¹²⁵I-hCG (α-subunit labeled) was used, indicating that the complex contains intact hCG. Upon reduction of the cross-linked receptor-¹²⁵I-hCG complex, a 105,000 mol wt complex in addition to the 130,000 one was observed. No smaller complexes appeared, however, upon reduction of the receptor-³H-hCG complex, suggesting that the α-subunit of hCG predominantly interacts with the receptor. The cross-linking efficiency was dependent on protein concentration, glutaraldehyde concentration, pH, reaction time and temperature. Under optimal conditions (2 mM glutaraldehyde, pH 7.0-8.0, 60 min, 20°C) no nonspecific complexes appeared and the efficiency of cross-linking of the partially purified detergent-solubilized receptor-¹²⁵I-hCG complex was approximately 30%. Glutaraldehyde thus provides a rapid and efficient cross-linking reagent to covalently attach ¹²⁵I-hCG to its receptor and is likely to be highly applicaple to other receptor-ligand systems as well.