B链氨端去－肽（B1）羧端去五肽（B26—30）猪胰岛素的初步晶体学研究

应用悬滴气相扩散法在含６％氯化钠和１２．５％丙酮的柠檬酸缓冲体系中,获得可供Ｘ射线结构分析用的Ｂ链氨端去－肽（Ｂ１）羧端去五肽（Ｂ２６－３０）猪胰岛素单晶体。晶体属四方晶系,空间群为Ｐ４１２２或Ｐ４３２２,晶胞参数为：ａ＝ｂ＝３６．０Ａ,ｃ＝１２０．０Ａ,α＝β＝γ＝９０度。单位晶胞中每个结晶学不对称单位含有２个ＤｅｓＢ１－ＤＰＩ分子。     

B链氨端去－肽（B1）羧端去五肽（B26—30）猪胰岛素的初步晶体学研究

应用悬滴气相扩散法在含６％氯化纳和１２．５％丙酮的柠檬酸缓冲体系中,获得可供Ｘ射线结构分析用的Ｂ链氨端去一肽（Ｂ１）羧端去五肽（Ｂ２６—３０）猪胰岛素（ＤｅｓＢ１－ＤＰＩ）单晶体。晶体属四方晶系,空间群为Ｐ４１２２或Ｐ４３２２,晶胞参数为：ａ＝ｂ＝３６．０Ａ,ｃ＝１２０．０Ａ,ａ＝β＝γ＝９０°。单位晶胞中每个结晶学不对称单位含有２个ＤｅｓＢ１—ＤＰＩ分子。     

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。     

高活力胰岛素研究：B3—Lys猪胰岛素的结晶及初步晶体学分析

用化学半合成方法制备的Ｂ３—Ｌｙｓ猪胰岛素（Ｂ３ＫＰＩ）具有与天然猪胰岛素相同的体内生物活力,但其体外生物活力及受体结合活力比天然猪胰岛素高。本文报道Ｂ３ＫＰＩ的晶体生长,初步晶体学分析及衍射数据与处理。Ｂ３ＫＰＩ晶体的衍射分辨率可以达到２．１Ａ（１Ａ＝０．１ｎｍ）,空间群为Ｒ３,晶胞参数为：ａＨ＝ｂＨ＝８２．２３ＡＣＨ＝３４．１７Ａ．收集了一套２．１Ａ分辨率的衍射数据,数据完全度为８１％,Ｒｍｅｒｇｅ为２．９％。     

高受体结合和低免疫原性的［B1—Ala，B2—Ala］－胰岛素

通过化学半合成从天然猪胰岛素得到［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］胰岛素。这一胰岛素类似物经聚丙烯酰胺凝胶电泳和ＨＰＬＣ鉴定证明是均一的,氨基酸组成与理论值相符生物活性测定结果表明：［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的体内活力与天然猪胰岛素相同,而与人胎盘细胞膜胰岛素受体的结合能力为天然猪胰岛素的１３２％。这一结果进一步说明胰岛素Ｂ链Ｎ端肽段参子与受体相互作用。此外,［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的免疫活性很低,远小于天然猪胰岛素的４％。     

家蚕病毒的表面增强拉曼光谱研究

得到并分析了家蚕核型多角体病毒（ＢｏｍｂｙｘｍｏｒｉＮｏｃｌｅａｒＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ缩写为ＢｍＮＰＶ）和质型多角体病毒（ＢｏｍｂｙｘｍｏｒｉｃｙｔｏｐｌａｓｍｉｃＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ缩写为ＢｍＣＰＶ）包涵体（即核型多角体ＢｍＮＰＢ和质型多角体ＢｍＣＰＢ）在银胶溶液中的表面增强拉曼散射（ＳＥＲＳ）光谱。ＢｍＮＰＢ和ＢｍＣＰＢ通过氨基酸残基侧链的Ｓ原子、ＣＯＯ－（ＣＯＯＨ）和ＮＨ２（ＮＨ）基团以及蛋白质分子的Ｎ－末端与银表面相作用。Ｔｒｐ残基金部或大部处于疏水环境中。ＢｍＮＰＢ中蛋氨酸残基侧链的Ｓ－ＣＨ２和ＣＨ２－ＣＨ２链分别为扭曲和扭曲式构型。ＢｍＣＰＢ中的Ｓ－ＣＨ２和ＣＨ２－ＣＨ２链则分别属于反式和扭曲构型;Ｃ－Ｃ－Ｓ－Ｓ－Ｃ－Ｃ链为反式－扭曲－反式结构。     

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）．     

百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

［B1－Ala，B2－Ala］－胰岛素：受体结合特性及生物活力研究

本文报道了［Ｂ１～Ａｌａ,Ｂ２－Ａｌａ］－胰岛素与人胎盘细胞膜胰岛素受体结合的特性和体外生物活力,并与胰岛素进行比较。在３７℃和杆菌肽存在下,１２５Ｉ－［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素和１２５Ｉ－胰岛素与人胎盘细胞膜作用依赖于反应时间,反应６分钟到达平衡,此时,［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素和胰岛素与胰岛素受体的最大结合分别为每毫克膜蛋白结合６．４４ｆｍｏｌ和３．４７ｆｍｏｌ：达到平衡一半所需时间（Ｔ１／２）分别为１９秒和２５秒。用１２５Ｉ－［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素作为放射配体进行竞争性结合研究,从ＩＣ（５０）得［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］胰岛素的受体结合活力为胰岛素的１３９．６％。Ｓｃａｔｏｈａｒｄ分析求得;［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素与高亲和和低亲和结合位点的结合常数在４℃时分别为５．８８×１０８Ｌ／ｍｏｌ和７．６３×１０５Ｌ／ｍｏｌ,而胰岛素分别为４．８３×１０８Ｌ／ｍｏｌ和３．３９×１０５Ｌ／ｍｏｌ。促脂肪细胞生成脂的实验表明：［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的活力为胰岛素的１３０％。     

去B链羧端七肽人胰岛素的分离纯化及性质研究

在大肠杆菌温度诱导体系中以非融合方式进行去Ｂ链羧端七肽人胰岛素原基因的表达,获得去Ｂ链羧端七肽人胰岛素原,表达产物占细胞总蛋白量的１３％,表达产物经ＳｅｐｈａｄｅｘＧ－５０柱层析分离及胰蛋白酶和羧肽酶Ｂ的酶促转化等步骤,可得到纯度达９４％以上的去Ｂ链羧端七肽人胰岛素,其氨基酸组成与预期值相符,受体活性是标准猪胰岛素的１％．     

Enzymatic semisynthesis of [LeuB30] insulin

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.     

Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in long-term treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low-cost platform for oral and injectable delivery of cleavable proinsulin.     

Synthesis and properties of carbonylbis(methionyl)insulin, a proinsulin analogue which is convertible to insulin by cyanogen bromide cleavage.

The preparation and use of carbonylbis (L-methionine p-nitrophenyl ester) as a reversible cross-linking reagent for insulin are described. The reaction of 1 equiv of reagent with zinc insulin in dimethylformamide in the presence of triethylamine yields as one of the products NalphaA1, NepsilonB29-carbonylbis(methionyl)insulin, (CBM-insulin). The CBM-insulin was characterized by end group analysis and by the products formed on tryptic and chymotryptic cleavage. It possessed 91% of the immunological and 6.5% of the hormonal activity of insulin. Treatment of CBM-insulin with cyanogen bromide (CNBr) in 70% formic acid for 1 h resulted in nearly complete removal of the methionine bridge to yield insulin. A small amount of a side product was removed on DEAE-cellulose at pH 7.2 to give an overall recovery of insulin of 70-80%. Oxidative sulfitolyses of CBM-insulin gave the hexa(S-sulfonate) which was reduced with dithiothreitol to yield reduced CBM-insulin. The latter compound, containing 6 sulfhydryls, exhibited a pH-dependent circular dichroic spectrum. The form at pH 10 exhibited a spectrum typical of random coil which was converted to a form at pH 7.8 which was characterized by a negative extremum at 213 nm. The change in the spectrum at 213 nm with pH was characterized by an apparent pKa of 8.5. Studies on the reoxidation of reduced CBM-insulin were performed at pH values between 7.8 and 10 and at protein concentrations of 0.01-1 mg/ml. The best yields (ca. 85%) of the correctly paired disulfide bonds were obtained in reoxidations at pH 9.5-10 at protein concentration of 0.01-0.1 mg/ml. CBM-insulin, which had been isolated from reoxidation at high pH of the reduced CBM-insulin, was cleaved by CNBr to yield a fully active insulin in an overall yield of 60% from the reduced CBM-insulin.     

Preparation of N,N-bis(methylsulphonylethoxycarbonyl)insulins (author's transl)]

The preparation of N,N-bis(methylsulfonylethoxycarbonyl)insulins is described. In an aequeous buffer at pH 5.8 selectivity of the reaction of insulin with 20 equivalents of N-(methysulfonylethoxycarbonyloxy)succinimide (Msc-ONSu) leads very specifically to N alpha A 1,-N alpha B 1-(Msc)2 - insulin. The product can be isolated in a yield of 60%. Using N alpha A 1-citraconylinsulin the N alpha B 1, NEB29-(Msc)2 -insulin can be prepared in a yield of 40% based on insulin.     

A new bifunctional reagent for the intramolecular crosslinking of insulin (author's transl)]

Reaction of bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone [SO2(Eoc-ONSu)2] with insulin in 1N NaHCO3/dimethylformamide forms NalphaA1,NepsilonA1,NepsilonB29-2,2'-sulfonylbis(ethoxycarbonyl)insulin [SO2(Eoc)2-insulin] in 20 - 35% yield. The product can be purified by partition chromatography. After cleavage of the disulfide bridges, reoxidation in very dilute solution reconstitutes about 60% of the original insulin activity. Cleavage of the crosslinking moiety can be achieved with 0.5N NaOH at 0 degrees C in only a few seconds, rendering a biologically fully active insulin.     

A shortened insulin with full in vitro potency

Des[(B26-30)-pentapeptide]insulin-B25-amide was prepared from protected des-[(B23-30)-octapeptide]insulin (pig) and H-Gly-Phe-Phe-NH2 by trypsin-mediated semisynthesis in a yield of 9% (based on insulin). The analogue was characterized with respect to chemistry, biological function and CD spectroscopy. While des[(B26-30)-pentapeptide]insulin with free carboxylate group exhibited a typical insulin activity of only 25% in vitro, des[(B26-30)-pentapeptide]insulinamide was fully active. Therefore des[(B26-30)-pentapeptide]insulin meets all structural and dynamic requirements for recognition and binding of the receptor as well as exertion of the biological effect, provided that the negative charge in the hydrophobic environment of PheB25 is neutralized.     

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.     

Intramolecular cross-linking of insulin. Preparation and properties of oxalyl- and malonyl-bis(methionyl) insulin

The bifunctional reagents, oxalyl-(Met-ONp)2 and malonyl-(Met-ONp)2 have been prepared and investigated as reversible cross-linking reagents for insulin and model compounds. The removal of the cross-linking residues was demonstrated by the cyanogen bromide cleavage of oxalyl-(Met-Phe-OMe)2 and malonyl-(Met-Phe-OMe)2. Zinc-insulin reacted with a molar equivalent of oxalyl-(Met-ONp)2 or malonyl-(Met-ONp)2 in presence of excess triethylamine to yield oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin, respectively. In these derivatives the N-terminal phenylalanine (B1 residue) was free. Thus the cross-link was between A1 and B29 residues in insulin. All three disulfide bonds of these insulin derivatives undergo reduction with tributylphosphine to give six sulfhydryls. Air-oxidation of reduced oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin in 0.05 M disodium phosphate, pH 9.5, yielded products which were indistinguishable from oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin respectively, as measured by physicochemical and biological methods. Cyanogen bromide cleavage of reduced and reoxidized malonyl-(Met)2-insulin in 70% formic acid regenerated insulin quantitatively, but only 40% of insulin was determined from similar treatment of oxalyl-(Met)2-insulin. The regenerated insulins exhibited the biological activity of native insulin. These studies strongly suggest that disulfide bonds formed during oxidation of reduced oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin are identical to those found in insulin.     

Sulfhydryl oxidation of reduced insulin in dilute solution

The reduction of insulin by tri-n-butylphosphine followed by air oxidation in dilute solution at pH 9.1 yields A- and B-chain disulfides. A(S-S)2 and B(S-S) have been purified on SP-Sephadex C-25 using a linear gradient of sodium chloride from 0.1 to 0.45 M in 0.5 M acetic acid containing 7 M urea. The overall yield of A(S-S)2 was 70%; and B(S-S), 60%. The A(S-S)2 and B(S-S) had the expected amino acid composition and N-terminal amino acid. The kinetics of reduction and reoxidation of insulin disulfide bonds are discussed.     

Processing and secretion of insulin-related peptides in an insulinoma cell line

Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line betaTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of betaTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed-phase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucose-rich growth medium the cells turned over their (pro)insulin inventory (90 +/- 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 muM) and isobutylmethylxanthine (1 mM), approximately 80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 +/- 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 muM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only approximately 50% converted to mature insulin, but 85 +/- 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 283-289, 1997.