比较不同类型白内障形成过程中非蛋白质巯基与蛋白质巯基的动态变化及关系

本文报道了在亚硒酸钠、平阳霉素及半乳糖诱发大鼠产生白内障过程中晶状体中非蛋白质巯基及蛋白质巯基的动态变化,并探讨了其变化机理及相互关系。在亚硒酸钠诱发白内障过程中,给药24h后晶状体中非蛋白质巯基减少到正常的二分之一,以后又逐渐回升,但始终未达到正常水平,至第7天,非蛋白质巯基又再度减少。在平阳霉素及半乳糖诱发白内障过程中,晶状体中非蛋白质巯基分别在给药后的第7天及第3天开始大量减少,以后继续减少,至第15天时,其含量分别为正常的十分之一及五分之一。在体外,亚硒酸钠有促进还原型谷胱甘肽自氧化的作用,半乳糖对此作用无影响,而平阳霉素可阻止其进行,但能加强亚硒酸钠的促进作用。在三种白内障晶状体中,蛋白质巯基开始减少的时间均较非蛋白质巯基为晚,这表明只有非蛋白质巯基减少到一定程度后蛋白质巯基才会被大量氧化,同时也说明非蛋白质巯基具有保护蛋白质巯基免受氧化的作用。只有这种保护作用减弱后,才会使蛋白质巯基遭受氧化而导致白内障。     

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.     

Introduction of sulfhydryl groups into proteins at carboxyl sites

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.     

Iron Increases Diabetes-Induced Kidney Injury and Oxidative Stress in Rats

Diabetic nephropathy is both a common and a severe complication of diabetes mellitus. Iron is an essential trace element. However, excess iron is toxic, playing a role in the pathogenesis of diabetic nephropathy. The present study aimed to determine the extent of the interaction between iron and type 2 diabetes in the kidney. Male rats were randomly assigned into four groups: control, iron (300-mg/kg iron dextran), diabetes (a single dose of intraperitoneal streptozotocin), and iron + diabetes group. Iron supplementation resulted in a higher liver iron content, and diabetic rats showed higher serum glucose compared with control rats, which confirmed the model as iron overload and diabetic. It was found that iron + diabetes group showed a greater degree of kidney pathological changes, a remarkable reduction in body weight, and a significant increase in relative kidney weight and iron accumulation in rat kidneys compared with iron or diabetes group. Moreover, malondialdehyde values in the kidney were higher in iron + diabetes group than in iron or diabetes group, sulfhydryl concentration and glutathione peroxidase activity were decreased by the diabetes and iron + diabetes groups, and protein oxidation and nitration levels were higher in the kidney of iron + diabetes group as compared to iron or diabetes group. However, iron supplementation did not elevate the glucose level of a diabetic further. These results suggested that iron increased the diabetic renal injury probably through increased oxidative/nitrative stress and reduced antioxidant capacity instead of promoting a rise in blood sugar levels; iron might be a potential cofactor of diabetic nephropathy, and strict control of iron would be important under diabetic state.     

Changes in the amount of nonhaem iron in the plasma, whole body, and selected organs during the postlarval life of the lamprey Geotria australis

The concentration of plasma nonhaem iron and the concentration and weight of all nonhaem iron in the whole body and selected organs, together with its partitioning into ferritin and haemosiderin iron, have been measured during the metamorphosis and upstream spawning migration of the Southern Hemisphere lamprey Geotria australis. Some nonhaem iron was lost from the animal during metamorphosis. However, the concentration and weight of nonhaem iron in the liver rose sharply at this time, following its release from important storage sites in adipose tissue and the degradation of larval haemoglobins. The nephric fold of larval and metamorphosing stages contained over 40% of all nonhaem iron in the body at the commencement of metamorphosis. This was predominantly in the form of haemosiderin. While the rise in liver iron during the transition from larva to adult primarily reflected an increase in the weight of ferritin iron, the amount of iron stored as haemosiderin rose conspicuously towards the end of metamorphosis. The rise in ferritin iron in the liver was accompanied by a decrease in ferritin iron in the plasma, which implies that changes in the liver during metamorphosis result in a greater filtering of circulating ferritin. Such a process would account for the very much lower plasma nonhaem iron concentrations which characterise later adult stages. The weight of nonhaem iron increased markedly in the liver and adult opisthonephros and in the whole animal during the nontrophic upstream spawning migration. This was primarily due to a marked rise in ferritin which in turn could be related to the degradation of adult haemoglobins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

Comparison of the action of streptomycin and neomycin on the structure of the bacterial ribosome

The influence of streptomycin and neomycin upon the conformation of the ribosome has been investigated using spin-labeled and fluorescent analogs of the sulfhydryl reagent, N-ethylmaleimide. Changes in the electron paramagnetic resonance spectra or in the polarization of fluorescence of labeled ribosomes reveal that streptomycin alters the mobility of labels bound to the sulfhydryl group of protein S18 while neomycin affects the mobility of labels bound to the sulfhydryl groups of proteins S1, S21 and/or L10. It is also observed that both streptomycin and neomycin interfere with changes in the mobility of labels induced by storage under inactivating conditions. From these results, it is concluded that: 1. streptomycin and neomycin distort the conformation of the ribosome at different sites, streptomycin disturbing preferentially the area around the sulfhydryl group of protein S18 while neomycin affects the environment of the sulfhydryl groups of proteins S1, S21 and/or L10; 2. streptomycin and neomycin interefere with the ability of the ribosome to undergo conformational changes.     

Synthesis and application of two reagents for the introduction of sulfhydryl groups into proteins

Two reagents are described which can be used for the introduction of sulfhydryl groups into proteins. Mercaptopropionylhydrazide modifies specifically periodate-oxidized N termini of proteins, provided that the N-terminal residue is serine or threonine. 3-(Phenyldithio)propionimidate introduces a disulfide bond at lysine residues of proteins. Reduction converts the disulfide into a sulfhydryl group. The imidate compound was found to react with a high specificity with only one lysine residue of ribosomal protein L7/L12.     

Sulfhydryl group reactivity in the Escherichia coli CoA transferase.

The acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli has the subunit structure α₂β₂ The enzyme contains six sulfhydryl groups, one per α chain and two per β chain, and no disulfides. The rates and extent of sulfhydryl group reactivity with 5,5′-dithiobis(2-nitrobenzoic acid) were compared in the free enzyme, the enzyme-CoA intermediate in the catalytic pathway, and a substrate analog-enzyme Michaelis complex. The analog used was acetylaminodesthio-CoA, a competitive inhibitor with respect to acetyl-CoA; the analog is not a substrate. The reactions were studied in the presence and absence of 10% glycerol. In the absence of glycerol, one sulfhydryl group reacted rapidly in the free enzyme and enzyme-CoA intermediate; relative to the free enzyme, the rate and number of subsequently reacting sulfhydryl groups were increased in the enzyme-CoA intermediate. In the presence of 10% glycerol, one sulfhydryl group reacted rapidly in the free enzyme, while two reacted rapidly in the enzyme-CoA compound; the rates and extents of subsequently reacting sulfhydryl groups were also enhanced in the enzyme-CoA compound. The data strongly suggested subunit interactions in the free enzyme and intermediate; glycerol abolished those interactions in the enzyme-CoA intermediate. In the absence of glycerol, sulfhydryl group reactivity in the Michaelis complex, enzyme-acetylaminodesthio-CoA, was similar to that in the free enzyme with one exception: One of the more slowly reacting sulfhydryl groups in the free enzyme reacted at a rate characteristic of the enzyme-CoA intermediate. The results obtained with N-ethylmaleimide were qualitatively similar. The fractional inactivation of the enzyme with N-ethylmaleimide as a function of sulfhydryl groups modified and the subunit location of those sulfhydryl groups indicated that the same sulfhydryl groups react in both enzyme species; however, those sulfhydryl groups reacted more rapidly in the enzyme-CoA compound. The data indicate both subunit interactions in the enzyme and characteristic conformational changes upon formation of an acyl-CoA-enzyme Michaelis complex and the enzyme-CoA intermediate.     

meso-α,ɛ-Diaminopimelate d-Dehydrogenase: Sulfhydryl Group Modification

meso-α,?-Diaminopimelate D-dehydrogenase was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and HgCl₂. Two sulfhydryl groups were titrated per molecule in the presence and absence of 6 M guanidine hydrochloride: the enzyme contained one sulfhydryl group per subunit. Modification of the sulfhydryl groups with p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-dithiopyridine, N-ethylmaleimide, and iodoacetic acid was accompanied by a loss of enzyme activity. However, modification of sulfhydryl groups of the enzyme with cyanide did not affect the activity. Thus, the introduction of bulky or charged substituents to sulfhydryl groups decreased the catalytic activity of the enzyme, but modification of the groups with the small and uncharged group, a cyano group, did not. The sulfhydryl groups did not play an essential role in catalysis.     

The influence of dietary iron on zinc in rat

The purpose of this study was to clarify the influence of iron on zinc status. The animals were divided into four groups, consisting of five rats in each group. The control group was fed on basal diet with adequate levels of zinc and iron, whereas the experimental group was fed diets containing different levels of iron ad libitum for 15 d. Low levels of iron (LFe) significantly increased the zinc absorption percentage but there was a decrease in high (HFe) and very high iron (VHFe) level groups (p<0.001). The retention percentage changes were found to be parallel to the changes in the absorption percentage curve. It was found that zinc (per total dry tissue) and Zn-65 (per total tissue) increased in the rats fed the LFe, whereas in general they decreased in the rats fed the HFe and VHFe diets. Significant changes were found in the duodenum and liver. Zn-65 (per g wet tissue) significantly increased in the brain and liver in the LFe group, but there was a decrease in the duodenum, ileum, kidney, liver, and brain in the HFe and VHFe groups. Changes in the level of zinc (per g dried tissue) were found to be parallel to the changes in Zn-65 in all the groups. The dietary proportions of iron appear to influence zinc metabolism at the intestinal and cellular transport levels over a given period of time.     

Levels of malondialdehyde production in rat liver following loading and unloading with iron

Rats were given daily injections of an iron sorbitol citric acid complex in a total dose of 50 mg Fe3+/100 g of body weight and either killed immediately after iron loading, or investigated 2 months later. Among the latter animals, one group was subjected to weekly phlebotomies in order to mobilize iron from the stores, while another group was not further treated. Quantitation of iron and malondialdehyde production was performed on homogenates of liver, kidney and spleen from controls and rats in the different experimental groups, and the distribution of iron in granular form was studied in the livers by means of electron microscopy. The results showed substantially increased amounts of iron in the organs studied after iron-loading and also augmented malondialdehyde production in the liver and kidney (but not in the spleen). A decreased malondialdehyde production was recorded two months after iron-loading in the kidney and spleen of non-bled animals; this decrease was exaggerated in the same organs from bled animals. The production of malondialdehyde as well as the iron content in the livers of both bled and non-bled rats 2 months after iron loading was higher than in the controls. The evidence obtained suggested that the accumulation of iron in the liver was causally related to increased lipid peroxidation. Judging from the morphological appearances this change did not result in cell damage, the only pertinent morphologic alteration being the occurrence of iron particles in the lysosomal vacuome and the cell sap.     

Influence of zymosan A on the content of ascorbic acid in mice

The aim of the study was to determine the effects of single intraperitoneal injections of zymosan A on changes in the content of ascorbic acid (ASC) in the brain, liver, spleen and kidneys of mature male mice, line Swiss. The experiments were carried out on 54 mice divided into 3 control groups and 6 experimental groups. Samples for analysis were collected after 3 h (experimental group I), 6 h (experimental group II) and 24 h (experimental group III) after the injection of zymosan A at the dose of 1 mg/kg body weight (b.w.). For groups IV, V and VI, the organs were removed at the same time as for the previous groups, but the animals were administered zymosan A at the dose of 100 mg/kg b.w. The content of ASC was then determined. The results showed that zymosan A significantly reduced the content of ASC in the brain of the mice in all the experimental groups, in the spleen in all the experimental groups except of group I (after 3 h since injection of zymosan A at 1 mg/kg b.w.), in the liver only in experimental groups IV, V and VI (after the injection of zymosan A at 100 mg/kg b.w.), while in the kidneys the effects were observed for groups III, V and VI. The data suggest that the observed decrease in the content of ASC is caused by the oxidative activity of zymosan A.     

Transferrin and Iron Uptake by the Brain: Effects of Altered Iron Status

Transferrin (Tf) and iron uptake by the brain were measured in rats using 59Fe-125I-Tf and 131I-albumin (to correct for the plasma content of 59Fe and 125I-Tf in the organs). The rats were aged from 15 to 63 days and were fed (a) a low-iron diet (iron-deficient) or, as control, the same diet supplemented with iron, or (b) a chow diet with added carbonyl iron (iron overload), the chow diet alone acting as its control. Iron deficiency was associated with a significant decrease and iron overload with a significant increase in brain nonheme iron concentration relative to the controls. In each dietary treatment group, the uptake of Tf and iron by the brain decreased as the rats aged from 15 to 63 days. Both Tf and iron uptake were significantly greater in the iron-deficient rats than in their controls and lower in the iron-loaded rats than in the corresponding controls. Overall, iron deficiency produced about a doubling and iron overload a halving of the uptake values compared with the controls. In contrast to that in the brain, iron uptake by the femurs did not decrease with age and there was relatively little difference between the different dietary groups. 125I-Tf uptake by the brains of the iron-deficient rats increased very rapidly after injection of the labelled proteins, within 15 min reaching a plateau level which was maintained for at least 6 h. The uptake of 59Fe, however, increased rapidly for 1 h and then more slowly, and in terms of percentage of injected dose reached much higher values than did 125I-Tf uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin.

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.     

三硝基甲苯对大鼠晶状体中可溶性蛋白质、巯基及二硫键含量的影响

我们采用三硝基甲苯（ＴＮＴ）与大鼠晶状体体外培养的方法,动态观察了晶状体中可溶性蛋白质、非蛋白质巯基、蛋白质巯基、蛋白质结合巯基及二硫键含量的变化,发现随着三硝基甲苯作用时间的延长,可溶性蛋白质、非蛋白质巯基及蛋白质巯基均减少,蛋白质结合巯基及二硫键交联的蛋白质含量增加,其中可溶性蛋白质、非蛋白质巯基及二硫键含量的变化皆达到了统计学上显著意义水平（Ｐ＜０．０５）。     

Characterization of Sulfhydryl Heterogeneity in Human Serum Albumin and Recombinant Human Serum Albumin for Clinical Use

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.     

Characterization of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.     

Inhibition of phosphate transport across the human erythrocyte membrane by chemical modification of sulfhydryl groups.

Effects of sulfhydryl-reactive reagents on phosphate transport across human erythrocyte membranes were examined using 31P NMR. Phosphate transport was significantly inhibited in erythrocytes treated with sulfhydryl modifiers such as N-ethylmaleimide, diamide, and Cu2+/o-phenanthroline. Quantitation of sulfhydryl groups in band 3 showed that the inhibition is closely associated with the decrease of sulfhydryl groups. Data from erythrocytes treated with diamide or Cu2+/o-phenanthroline demonstrated that intermolecular cross-linking of band 3 by oxidation of a sulfhydryl group, perhaps Cys-201 or Cys-317, decreases the phosphate influx by about 10%. The inhibition was reversed by reduction using dithiothreitol. These results suggest that sulfhydryl groups in the cytoplasmic domain of band 3 may play an important role in the regulation of anion exchange across the membrane.     

Effect of sulfhydryl compounds on the automatism of the pacemakers]

Experiments were performed on the isolated rabbit hearts and also on the hearts with complete atrioventricular block; a study was made of the effect of an excess or deficiency of the sulfhydryl groups on the automatism of the cardiac pace makers. Unithiol and cysteine in concentrations of 1-10(-6)-1-10(-4) g/ml were used as donors of sulfhydryl groups; deficiency of these groups was induced by the alloxan administration in concentrations of 1-10(-5)-5-10(-5) g/ml. Changes in the sulfhydryl group content produced no marked effect on the automatism of the synoatrial node. An excess of sulfhydryl groups promoted poststimulation depression of the automatism of the potential pace makers of the cardiac ventricles and could lead to the origination of Luciani's periods. On the contrary, in case of a deficiency of the sulfhydryl groups there was a sharp elevation of the automatism of the ventricular pace makers, the atrioventricular conduction became disturbed, and the poststimulation depression of the automatism became considerably diminished. Disturbances of the cardiac activity caused by the sulfhydryl group deficiency were completely eliminated by unithiol or cysteine.