Location of SH groups along the polypeptide chain of soybean beta-amylase

The five SH groups of soybean beta-amylase differ in reactivity toward SH reagents such as 2,2'-dithiopyridine (2-PDS), monoiodoacetate and N-ethylmaleimide (NEM). They were designated as SH1, SH2, SH3, SH4, and SH5, in order of their reactivity except for the two buried SH groups, SH4 and SH5. The location of the five SH groups along the polypeptide chain was determined by specific cleavage at the amino side of their cyanocysteine residues which were formed by converting SH to SCN groups by cyanide after modifying the SH groups with 2-PDS. The selective modification of SH groups was achieved as follows: SH1 reacted with 2-PDS at low and high ionic strength, while SH2 reacted only at high ionic strength. SH2 and SH3 were also modified with 2-PDS using SH1-carboxymethylated soybean beta-amylase. The buried SH groups, SH4 and SH5, were modified with 2-PDS under the denaturation conditions after the reactive SH groups, SH1, SH2, SH3, were irreversibly blocked with NEM. On the other hand, the five SH groups were cyanylated with [14C]cyanide or with 2-nitro-5-thiocyanobenzoic acid (NTCB) for the cleavage at all five SH groups. The molecular weight estimation of derivatives of cleaved soybean beta-amylase by SDS-gel electrophoresis showed that the five pairs of fragments (Mw 50,000 & 6,500, 47,000 & 8,000, 38,000 & 18,000, 35,000 & 23,000, and 31,000 & 25,000) were identified with the fragments formed by cleavage at SH1, SH2, SH3, SH4, and SH5, respectively. By considering fragments incorporating 14C (Mw 47,000, 35,000, 25,000, 18,000, and 6,500), the fragments were aligned along the polypeptide chain of soybean beta-amylase, in order from the N-terminus as SH2, SH5, SH3, SH4, and SH1. This order is supported by estimating the molecular weight of fragments formed by high-yield cleavage using NTCB and by analyzing the COOH-terminal residues of the fragment cleaved at SH2.     

Oxidation of reactive sulfhydryl groups of sarcoplasmic reticulum ATPase

The role of reactive sulfhydryl groups of sarcoplasmic reticulum ATPase has been investigated. Incubation of ATPase with 17 mol o-iodosobenzoic acid per mol ATPase results in a 15% inhibition of Ca2+ uptake with only a 5% loss of ATPase activity. When ATPase is treated with 15 mol KMnO4 per mol ATPase, Ca2+ uptake is completely inhibited. From the measurement of remaining SH groups using 5,5'-dithiobis-(2-nitrobenzoic acid), it is found that the oxidation of approximately four SH groups per ATPase molecule with KMnO4 leads to a complete loss of Ca2+ uptake, while the oxidation of five SH groups per ATPase with o-iodosobenzoic acid results in only 15% inhibition of Ca2+ uptake. The results of amino acid analysis indicate that KMnO4 oxidizes the reactive SH groups to sulfonic acid groups. Among the five o-iodosobenzoic acid-reactive SH groups, at least one shows a distinct Ca2+ dependence. Addition of o-iodosobenzoic acid to the reaction medium containing KMnO4 does not increase the number of oxidized SH groups, indicating that both o-iodosobenzoic acid and KMnO4 oxidize the same SH groups of the enzyme. The different effects of two oxidizing agents on sarcoplasmic reticulum ATPase eliminate the possibility of direct involvement of SH group(s) in the ATPase reaction.     

Urinary oligosaccharides of GM1-gangliosidosis. Structures of oligosaccharides excreted in the urine of type 1 but not in the urine of type 2 patients

Among the seven oligosaccharide fractions obtained by Bio-Gel P-4 column chromatography of urine of GM1-gangliosidosis Type 1 patients, three fractions (peaks V, VI, and VII) were completely missing in the urine of GM1-gangliosidosis Type 2 patients. Structural study of these oligosaccharide fractions by sequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation has shown that peaks V, VI, and VII are mixtures of 16, 30, and 49 isomeric oligosaccharides. All these 95 oligosaccharides contain Gal beta 1 leads to 4GlcNAc beta 1 lead to 3 repeating structures in their outer chain moieties, indicating that the tissues of GM1-gangliosidosis Type 2 patients do contain beta-galactosidase activity which releases readily galactose residue from such repeating sugar chains.     

The order of cyanogen-bromide peptides and location of carbohydrate in the alpha2 chain of guinea-pig skin collagen.

The alpha2 chain of guinea pig skin collagen contains two additional methionyl residues in comparison with the alpha2 chain of other vertebrate species. The order of the three CNBr peptides unique to the alpha2 chain was established on the basis of the homology of their primary structures to sequences of previously ordered regions in the alpha1 and alpha2 chains of other colagens. The two larger peptides, 4A + 4B, were found to correspond to the region homologous to alpha2-CB4 of other species, while the smaller peptide, 3A, was homologous to the NH2-terminal portion of alpha2-CB3. Thus, the order of the peptides in the alpha2 chain of this collagen is 1-O-4A-4B-2-3A-3B-5. Periodate oxidation and alkaline or acid hydrolysis of the CNBr fragments showed that all of the hydroxlysine-linked carbohydrate in the alpha2 chain was present in alpha2-CB4B. Carbohydrate analyses were most consistent with the existence of single monosaccharide and disaccharide units in this region.     

反相高效液相层析在胰岛素结构与功能研究中的应用

采用一种简单的“甲醇-水-0.1％三氟醋酸”作为洗脱体系的反相高效液相层析对研究胰岛素结构与功能关系,以及A链和B链相互作用时所用的一些胰岛素衍生物进行了快速、灵敏、准确的分析和鉴定。 胰岛素的S-磺酸型A链和B链只经一次离子交换层析纯化,经鉴定结果表明是均一的。S-硫甲基型A链和B链基本上也是均一的。通过对胰岛素还原产物的分析确定了能保证胰岛素折分完全并对其重组较为有利的还原条件。去B链羧端五肽(B26—30)胰岛素,以及去B链羧端八肽(B23—30)胰岛素能同时与天然胰岛素清楚地分离。一级结构差别极小的猪胰岛素和牛胰岛素也可得到分辨。胰岛素的羧端缩短的B链和A链重组的定量分析表明B链羧端肽段不但对胰岛素分子的生物活力的体现,而且在A链和B链的正确重组中都起着重要的作用。     

FTIR studies of secondary structures of bovine insulin and its derivatives

The amide I bands of the deconvolved FTIR spectrum of bovine insulin, despentapeptide (B26-B30) insulin and desoctapeptide (B23-B30) insulin in D2O solution have been assigned to alpha-helix, the 3(10) helix, irregular helix, extended chains, beta-turns and other secondary structures. From the peak areas the relative contents of these structures obtained are in general agreement with those calculated from the known structures of porcine insulin and DPI in the crystalline state. The main difference in the structure of DOI with those of insulin and DPI is the shortening of the helix segment and an extended chain for the C terminal segment in the B chain.     

Photocross-linking from SH1 of the myosin heavy chain to light chains through the dinitrophenyl group attached to SH1

Trinitrobenzene selectively dinitrophenylates SH1, a specific thiol in the myosin heavy chain which contains 1 mol of this cysteinyl residue. When the SH1-DNP-myosin thus obtained was irradiated with a mercury lamp, a cross-linked product was formed with a molecular weight of about 220K daltons. It was shown that this product was composed of both heavy and light chains by fluorescence labeling of the heavy chain at SH2, another specific thiol, and immune reaction using an anti-light chain antibody, respectively.     

Formation and splitting of esters in subterminal oxidation of dodecane by Fusarium lini

Summary Extracellular oxidation products having the same number of carbon atoms as the alkane that was oxidized were isolated from a Fusarium lini culture broth grown on n-dodecane. They were secondary isomeric alcohols, corresponding isomeric ketones and isomeric esters with 12 carbon atoms.Esterase activity in cell-free extracts of the fungus which was incubated on a p-nitrophenyl-acetate substrate increased with increasing temperatures and pH-values in the ranges 20–40°C and pH 6.0 to 8.0 respectively. The activity, when incubated on p-nitrophenyl-acetate,-laurate and-palmitate substrates, decreased with decreasing fatty acid chain lengths. When incubated with isomeric esters consisting of 12 carbon atoms, it was influenced by the ester linkage position in the chain. When the alcohol chain length in the ester increased from one to six carbon atoms, the esterase activity decreased. The same effect was observed when the chain length of the acid increased from two to six carbon atoms. Minimum esterase activity was reached when both the alcohol and the acid had a chain length of six carbon atoms.The view that all ketones produced during subterminal oxidation of alkanes by Fusarium lini and perhaps other members of Moniliales are further metabolized via ester intermediates is supported. A probable non-specific esterase or lipase catalyses the hydrolysis of the isomeric esters which are formed from the ketones.     

Structural study of long arm fragments of laminin. Evidence for repetitive C-terminal sequences in the A-chain, not present in the B-chains

The outer segments of the long arm of laminin have recently been shown to mediate attachment of many cell types and to stimulate neurite outgrowth. For a structural characterization of this part of the molecule we prepared, by limited elastase digestion of laminin, fragments E3 and E8, previously identified as a globular heparin-binding domain and as a 35-nm-long rod with a terminal globule, respectively. Fragment E3 is a domain adjacent to fragment E8. Both structures together comprise the complete terminal half of the long arm. Our data confirm current models, which predict that the C-terminal segments from all three chains contribute to its structure. The B chains terminate at the end of the rod like domain, while the large terminal globule is formed by A-chain structures only. In addition to fragment E3, two new fragments T1 and T2 obtained by tryptic cleavage of fragment E8 were characterized as substructures of the globular domain. Screening of a mouse cDNA library with synthetic oligonucleotides allowed isolation of an 1.8-kb cDNA clone encoding 547 C-terminal amino acids of the A chain and some 196 nucleotides of the 3'-untranslated region including a single polyadenylation site. The clone contained portions of domain T2 and the complete heparin binding domain E3 which was thus identified as the most C-terminal domain of the A chain. Sequence alignment indicated that the terminal globule is formed by homologous repeats of some 140 residues having no counterpart in the B chains.     

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.     

Reduction of S-sulpho groups by tributylphosphine: an improved method for the recombination of insulin chains.

All 4 S-sulpho groups of the S-sulpho substituted insulin A-chain could be removed with 4 mol of tributylphosphine. Reduction of a 1:1 mixture of both S-sulpho insulin chains with tributylphosphine followed by air oxidation gave insulin which was isolated in pure form and high yield. Removal of excess reducing agent was not necessary, in contrast to the usual procedures employing thiols for the reduction step. This constitutes a rapid and simple method for the generation of insulin from its chains. A new method for the purification of S-sulpho-A-chain has been developed.     

<Emphasis Type="Italic">In vitro</Emphasis> insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.0 to 8.0 causes a reduction in the apparent molecular weight of the enzyme at high KCl concentration, which can be interpreted as due to a deprotonation-induced isomerization process. Removal of Zn2+ from AMP deaminase has effect similar to alkalinization in modifying the sedimentation properties of the enzyme. In the native enzyme at high K+ concentration about 7, 9 and 12 SH groups can be titrated with Nbs2, approximately 1, 2 and 4 SH groups reacting as fast sets, at pH 6.0, 7.0 and 8.0, respectively. Substitution of the 12 SH groups reactive with Nbs2 at pH 8.0 has no effect on the pH-dependent allosteric behaviour of the enzyme. Removal of K+ causes considerable changes in the reactivity of AMP deaminase towards Nbs2, unmasking a class of additional SH groups, so that the total number of titratable SH groups approaches that of 30 determined in denaturing conditions. In the enzyme previously treated with N-ethylmaleimide to alkylate the fast reacting class of SH groups, the class of additional SH groups are substituted by Nbs2 at basic pH, but not at acidic pH, with a concomitant reduction of the enzyme activity.     

In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient Ⅰ was formed from P3A through thiol/disulfide exchange reaction; then, transients Ⅱ and Ⅲ, each containing two native disulfides, were formed through the recognition and interaction of transient Ⅰ with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients Ⅱ and Ⅲ, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma. Appearance of triantennary sugar chains and unique biantennary sugar chains

Human chorionic gonadotropin (hCG) highly purified from urine of the patient with choriocarcinoma contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The structures of these sugar chains were determined by the combination of sequential glycosidase digestion, periodate oxidation, and methylation analysis. As compared with the sugar chains of normal urinary and placental hCG reported previously, they include several prominent structural differences. More than 97% of the sugar chains of choriocarcinoma hCG was free from sialic acid, while the sugar chains of normal hCG were mostly sialylated. Choriocarcinoma hCG contains unusual biantennary complex-type sugar chains in addition to regular tri-, bi-, and monoantennary sugar chains. These sugar chains have two outer chains linked at the C-2 and C-4 positions of the same alpha-mannosyl residue of the trimannosyl core. Since normal hCG does not contain any triantennary sugar chains, occurrence of Gal beta 1 leads to 4GlcNAc beta 1 leads to 4Man alpha 1 leads to group is another characteristic feature of the sugar chains of choriocarcinoma hCG. The evidence that the monoantennary sugar chain of Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc leads to Asn is not found in normal hCG and the sum total of fucosylated sugar chains is 50%, which is twice as much as normal hCG, indicated that fucosylation is also modified in choriocarcinoma tissue.     

S-硫甲基型胰岛素A及B链相互作用的200MHz~1H NMR研究

前已报道,S-硫甲基型胰岛素A及B链相混后,α螺旋等有序二级结构含量增加,部分酪氨酸残基由相对亲水的环境逐渐转移到相对疏水的环境,整个过程在1.5～2小时达到稳定,说明在还原型A、B链能重组成有活力的胰岛素的相同条件下,两肽链间确实存在着次级键相互作用。本文报道以200兆赫NMR~1H谱所测S-硫甲基型A及B链的图谱,对其主要谱峰作了初步识别。在pH10.8时,A及B链以克分子比1.5:1相混后,Tyr的(2,6)位及(3,5)位质子峰较之单独链有低场位移,提示Tyr的酚羟基解离减少,Tyr转移至相对疏水的环境;也可能是肽链构象变化,使Tyr受去屏蔽效应所致。在A、B链相混后,每半小时累加的结果表明,不仅Tyr的两个低场峰随时间有低场位移,甲基峰随时间也有连续的低场位移,在1.5小时后达到稳定。此时间过程与前者报道的紫外差光谱、园二色性的观察结果一致,说明构象的调整涉及整个肽链。不同pH下的~1H谱测定显示,在一段pH区间都存在着A、B链间的次级键相互作用。上述结果表明,或许正是这种链间的次级键相互作用,才使A、B链之巯基有可能正确配对,重组成有生物活力的胰岛素分子。     

Modification of alpha-crystallin subunit chains and formation of alpha-neoprotein molecules: role of SH groups

Immunoadsorbents with bound antibodies restricted to determinants dependent on alpha-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled alpha-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on alpha-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified alpha-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fast-reacting surface SH groups. (ii) The modified alpha-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in alpha-crystallin, indicating formation of alpha-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming alpha-neoprotein molecules.     

Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.     

还原型胰岛素A、B链相互作用的初步研究

本文报道在chance所用“一步法”重组胰岛素的类似条件下,还原型A、B链相互作用研究的初步结果。在pH10.6对S—磺酸型A及B链加入1.2倍SH/SSO_3—的二巯基苏糖醇,在一小时内A、B链与DTT的混合物较之单独的A、B链与DTT的混合物,紫外差光谱显示295、245nm负差峰,峰值随时间增大;园二色性测定观察到混合链的α螺旋含量比单独的A、B链有所增加。这表明在A、B链的巯基氧化生成二硫链前,还原型A、B链间的相互作用导致Tyr残基逐渐内埋,肽链构象的调整使有序二级结构增加。根据此时还原程度的测定,讨论了DTT与S—磺酸型A、B链作巯基交换的可能机制,过多的还原试剂易破坏已正确配对的二硫键,也妨碍肽链构象的调整。