王不留行环肽研究

从中药王不留行（Ｖａｃａｒｉａｓｅｇｅｔａｌｉｓ）种子中分离并鉴定了４个环肽化合物,分别命名为王不留行环肽Ａ,Ｂ,Ｃ,Ｄ（ｖａｃｃａｒｉｎｓＡ,Ｂ,Ｃ,Ｄ）,其中王不留行环肽Ａ为新环肽化合物。其结构通过光谱和化学方法分别确定为：ｖａｃｃａｒｉｎＡ——ｃｙｃｌｏ－（Ｔｒｐ－Ａｌａ１－Ｇｌｙ－Ｖａｌ－Ａｌａ２）,ｖａｃｃａｒｉｎＢ———ｃｙｃｌｏ－（Ｐｒｏ－Ｇｌｙ－Ｌｅｕ－Ｓｅｒ－Ｐｈｅ１－Ａｌａ－Ｐｈｅ２）,ｖａｃｃａｒｉｎＣ———ｃｙｃｌｏ－（Ｐｒｏ１－Ｇｌｙ－Ｔｙｒ－Ｖａｌ－Ｐｒｏ２－Ｌｅｕ－Ｔｒｐ）,ｖａｃａｒｉｎＤ———ｃｙｃｌｏ－（Ｐｒｏ－Ｖａｌ１－Ｔｒｐ－Ａｌａ－Ｇｌｙ－Ｖａｌ２）．     

瓦草中三个新环肽

从云南民间中草药瓦草（Ｓｉｌｅｎｅｓｚｅｃｈｕｅｎｓｉｓ）的根中分离鉴定了３个新的环肽,分别命名为瓦草环肽Ａ,Ｂ,Ｃ（ｓｉｌｅｎｉｎｓＡ,Ｂ,Ｃ）用光谱和化学方法确定它们均为环八肽,其结构分别为：ｓｉｌｅｎｉａＡ－ｃｙｃｌｏ－（Ｐｒｏ－Ｌｅｕ－Ｓｅｒ－Ｐｈｅ－Ｐｒｏ－Ｔｙｒ－Ｌｅｕ－Ｖａｌ）,ｓｉｌｅｎｉｎＢ－－ｃｙｃｌｏ－（Ｐｈｅ－Ｌｅｕ－Ａｌａ－Ｐｒｏ－Ｌｅｕ－Ｐｒｏ－Ｐｈｅ－Ｐｒｏ）,ｓｉｌ     

千针万线草环肽A的结构修正

从云南民间药物千针万线草（Ｓｔｅｌｌａｒｉａ ｙｕｎｎａｎｅｎｓｉｓ）的根中分离到一新环肽ｓｔｅｌｌａｒｉｎＡ。前报主要利用快速原子轰击质谱确定其结构为ｃｙｃｌｏ（Ｇｌｙ－Ｐｒｏ－Ｐｈｅ－Ｔｙｒ－Ｇｌｙ－Ｇｌｙ－Ｐｒｏ）。最近利用最新二维核磁结合快速原子轰击质谱及酶降解重新考察了其结构,修正为ｃｙｃｌｏ（Ｇｌｙ－Ｐｒｏ－Ｐｈｅ－Ｐｒｏ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ）。     

双链杂交分子“胰岛素－类胰岛素生长因子－I

以去Ｂ链Ｃ端八肽胰岛素（ＤＯＩ）和化学合成的ＩＧＦ－Ｉ的２２～２９（８肽）及２２－３２（１１肽）为底物,用酶促半合成方法制备了杂交分子“胰岛素－类胰岛素生长因子－Ｉ,Ｉｎｓ／ＩＧＦ－Ｉ（８）和Ｉｎｓ／ＩＧＦ－Ｉ（１１）。研究了它们的胰岛素生物活性,结果表明,猪胰岛素Ｂ链Ｃ端Ｂ２７的Ｔｈｒ被Ａｓｎ取代,Ｂ３０的Ａｌａ被Ｔｈｒ取代同时Ｂ２５～Ｂ２６及Ｂ２８－Ｂ２ｋ９氨基酸顺序颠倒以及在Ｂ链Ｃ末端延长３肽（Ｇｌｙ－Ｔｙｒ－Ｇｌｙ）都不影响胰岛素的生物活力。     

凤眼莲根分泌物氨基酸组分对根际肠杆菌属F_2细菌的趋化作用

凤眼莲（Ｅｉｃｈｈｏｒｎｉａｃｒａｓｓｉｐｅｓ）的根分泌物中含有Ｍｅｔ等多种氨基酸,其中Ｍｅｔ、ＧＡＢＡ、Ｇｌｙ、Ａｌａ、Ａｓｐ、Ｓｅｒ、Ｖａｌ和Ｌｅｕ（１０－７～１０－２ｍｏｌ·Ｌ－１）均对凤眼莲的根际肠杆菌属Ｆ２（Ｅｎｔｅｒｏｂａｃｔｅｒｓｐ．Ｆ２）细菌有强烈的正趋化作用;Ｇｌｕ、Ｔｈｒ和Ｈｉｓ（１０－７～１０－３ｍｏｌ·Ｌ－１）也对该菌有一定的正趋化作用;而Ｌｙｓ、Ｃｙｓ、Ａｒｇ、Ｔｙｒ、Ｐｒｏ、Ａｓｎ、Ｇｌｎ、Ｉｌｅ、Ｐｈｅ和Ｔｙｐ则对该菌表现出一定的负趋化作用．对细菌的正趋化作用存在一个趋化物的最适浓度范围．具有正趋化作用的氨基酸在凤眼莲根际的浓度都较高,而具有负趋化作用的浓度则较低,这正是凤眼莲与该根际细菌结合为根际微生态系统的原因之一．     

大鼠甘氨肽酰化单氧酶在CHO细胞中的活怀表达及在体外酰胺化？…

将大鼠脑ＣＤＮＡ库来源的ＰＡＭ基因片段,经克隆重组,成真核表达质粒ＰＳＶ－ＰＡＭ〈并转染ＣＨＯ细胞,获得在ＣＨＯ中稳定表达活性型α－酰胺化酶的细胞株ＤＧＡＥ。 物为双功能酶,分泌至培养基中的酶活力远高于胞内。体外酰胺化加工研究表明。以α－Ｎ＿ａｃｅｔｙｌ－Ｔｙｒ－Ｖａｌ－Ｇｌｙ为底物,该酶催化反应的Ｋｍ为１２．５μｍｏｌ／Ｌ,Ｖｍａｘ为１８０μｍｏｌ／ｍｇ／ｈ,而且催化反应中表现中有最适铜离子浓度     

〔B25－Trp〕去B链C端五肽胰岛素酰胺的合成及性质

去Ｂ链Ｃ端八肽胰岛素（ＤＯＩ）与Ｇｌｙ－Ｐｈｅ－Ｔｒｐ－ＮＨ２通过胰蛋白酶催化缩合后,经分离纯化得到聚丙烯酰胺凝胶电泳均一的Ｂ２５－Ｔｒｐ取代的去Ｂ链Ｃ端五肽胰岛素酰胺（〔Ｂ２５－Ｔｒｙ〕ＤＰＩ－ＮＨ２）．酶促缩合率大于８５％,回收率为３６．２％。用小白鼠惊厥法测得〔Ｂ２５－Ｔｒｐ〕ＤＰＩ－ＮＨ２的体内活力约为天然胰岛素的７０％,用人胎盘细胞膜测得的其胰岛素受体结合能力为天然胰岛素的７２．５±２．４％;利用色氨酸和酪氨酸的内源荧光研究了此类似物的溶液构象．     

DCM和DMF对DCC—HOBt系统催化酯化及酰化反应的影响

ＤＣＣ－ＨＯＢｔ系统在ＤＣＭ和ＤＭＦ溶剂中,催化Ｆｍｏｃ－Ｌｅｕ－ＯＨ与王氏树脂的酯化反应,其结合率分别为１８％和１０％,该系统催化Ｆｍｏｃ－Ｐｈｅ－ＯＨ与Ｈ２Ｎ－Ｌｅｕ－＝ｒｅｓｉｎ的酰化反应中,溶剂为ＤＣＭ时,反应３０ｍｉｎ,其结合率为１００％;当ＤＣＭ：ＤＭＦ体积比为１：３．５时,反应１３０ｍｉｎ,其结合率为８１％,表明ＤＣＣ－ＨＯＢｔ系统宜在ＤＣＭ非极性溶剂论肽键的形成。     

金铁锁根中的环肽成分

从云南民间重要的药用植物金铁锁（Ｐｓａｍｍｏｓｉｌｅｎｅ ｔｕｎｉｃｏｉｄｅｓ Ｗ．Ｃ．Ｗｕ ｅｔ Ｃ．Ｙ．Ｗｕ）的根中分离得到２个新的天然环二肽以及２个新的环八肽：金铁锁环肽Ａ和Ｂ（ｐｓａｍｍｏｓｉｌｅｎｉｎｓ Ａ ａｎｄ Ｂ）。它们的结构经光谱方法鉴定为ｃｙｃｌｏ（－Ａｌａ－Ａｌａ－）,ｃｙｃｌｏ（－Ｖａｌ－Ａｌａ－）,ｃｙｃｌｏ（－Ｐｒｏ１－Ｐｈｅ１－Ｐｒｏ２－Ｐｈｅ２－Ｐｈｅ３－Ａｌａ－ｐ     

蜈蚣碱性蛋白SSmp-d的分离纯化及其部分理化性质的鉴定

少棘巨蜈蚣（ＳｃｏｌｏｐｅｎｄｒａｓｕｂｓｐｉｎｉｐｅｓｍｕｔｉｌａｎｓＬ．Ｋｏｃｈ）经９５％乙醇脱脂后,再经４℃水冷渗,水提液低温旋转浓缩,冻干,得到的冻干粉先后经过ＳｅｐｈａｄｅｘＧ－２５柱,等电聚焦制备电泳,再经ＳｅｐｈａｄｅｘＧ－１５０柱,ＳｅｐｈａｄｅｘＧ－１００柱,最后经ＨＰＬＣ制备得到一个纯的碱性蛋白,命名为ＳＳｍｐ－ｄ．该蛋白经ＨＰＬＣ、超薄等电聚焦电泳检验是均一的．采用ＨＰＬＣ和Ｐｒｏｔｅｉｎ－ＰａｋＴＭ１２５柱测定其分子量为２４．６４ｋＤ．ＩＥＦ－ＨＰＣＥ显示其等电点为９．２７．氨基酸分析表明ＳＳｍｐ－ｄ含较多的Ａｒｇ、Ｌｙｓ等碱性氨基酸,另外还含有较多的Ａｌａ、Ｌｅｕ．使用蛋白质自动序列分析仪测定了ＳＳｍｐ－ｄＮ端的１１个氨基酸,序列为ＮＨ３＋－Ａｓｐ－Ｖａｌ－Ａｓｎ－Ｐｈｅ－Ａｒｇ－Ｌｅｕ－Ｓｅｒ－Ｇｌｙ－Ａｌａ－Ａｓｐ－Ｐｒｏ．     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Isolation and structure identification of a morphine-like peptide "enkephalin" in bovine brain.

The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides HTyrGlyGlyPheLeuOH (Leucine-enkephalin) and HTyrGlyGlyPheMetOH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.     

Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, buried in the dimer of insulin, were identified by means of alanine-scanning mutagenesis. The receptor binding activity, in vivo biological potency and self-association properties of the seven single alanine human insulin mutants were determined. Four of the seven single alanine mutants, [B12Ala]human insulin, [B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]human insulin, are monomeric insulin, which indicates that B12Val, B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulin dimer. The monomeric [B16Ala]human insulin and [B26Ala]human insulin retain 27 and 54% receptor binding activity, respectively, and nearly the same in vivo biological potency compared with native insulin, so they could be developed as the fast-acting insulin.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

News & Notes: Specificity of a Neutral Zn-Dependent Proteinase from Thermoactinomyces sacchari Toward the Oxidized Insulin B Chain

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms. Received: 10 November 1999 / Accepted: 15 December 1999     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.