Synthesis of trypsin inhibitor CMTI III from squash seeds (Cucurbita maxima)

Summary Using the solid-phase procedure, a 29-peptide cross-linked by three disulphide bridges was synthesized. The synthetic product was shown to be identical with the trypsin inhibitor CMTI III from squash seeds (Cucurbita maxima).Acknowledgments. This work was supported by the Swedish Natural Science Research Council (K-GF 3020-107) and the Polish Academy of Sciences (Research grant No. MR I 12.1.6.1).     

Multiplicity of beta lactamases: a problem of isoenzymes]

Analytical isoelectric focusing of beta lactamases is a very sensitive and powerful technique which generally shows a number of satellites near the main enzymatic spike. By this technique we studied two bacterial groups known to produce different beta lactamases, but only one by germ. Analytical isoelectric focusing showed that all bacteria from the same group produce the same satellite system which differs only in relative activity.     

Problems in the determination of isoelectric points of beta lactamases]

Analytical isoelectric focusing in polyacrylamide gels and preparative electrofocusing in density gradients may give very different pI determination for the same beta lactamase. This difference seems due to a lack of enzyme migration in some parts of the cross-linked polyacrylamide gel. This migration appears easier in density gradient isoelectricfocusing, and thus this technique yields more significant absolute pI values. Analytical isoelectric focusing should only be used for comparison studies.     

Isozymes of cathepsin B1 in developing human placenta

Summary Cathepsin B1 was purified from human placentas of different gestational ages and analyzed by isoelectric focusing on polyacrylamide gel. The enzyme was shown to consist of 3 or 4 isozymes with pI values between 5.1 and 5.7.     

Different primary structure of 2 variants of Friend virus p 30 polypeptide separated by isoelectric focusing]

Variants of p 30 (iso-p 30s), the 30,000 dalton major polypeptide of murine C-type retraviruses, have been characterized in all virus strains by isoelectric focusing. Several of these iso-p 30s have been found to coexist in a given virus strain. In the present study, two iso-p 30s, characteristic of the Friend-Rauscher subgroup, separated by preparative isoelectric focusing of p 30 in thin layers of polydextran gel, were subjected to tryptic peptide mapping and aminoacid analysis. The two iso-p 30s, with isoelectric points (pI) of 6.5 and 7.1, were found to possess a partially different primary structure, with about 50% homology. The results are discussed.     

Isoelectric variants of the principal polypeptide (p 30) of different strains of murine oncornaviruses]

The major murine oncornavirus polypeptide p 30 (30,000 daltons), which bears the conventional group-specific (gs) determinants, can be separated by isoelectric focusing into four classes, with isoelectric points (pI) of 5.5, 5.9, 6.7 and 7.1. All the Moloney stocks tested have a pI 5.9 p 30, while p 30's with three to four different pI values are found to coexist in other stocks, with the pI 6.7 form predominating in Gross virus stocks.     

Genetic variation in esteroproteases in the mouse submandibular gland

Summary 9 isozymes of esteroproteases were detected by column isoelectric focusing of submandibular gland extracts from four inbred strains of male mice. A marked strain variance in the esteroprotease isozymes was found among the strains.     

Some properties of the goat placental lactogen.

Goat placental lactogen was partially purified from a medium collected after placental tissue incubation. The data obtained by disc electrophoresis and isoelectric focusing experiments, as well as by means of radioreceptor assay methods, provide evidence of the similarity between the goat and ovine placental lactogen.     

Isolation and characterization of biologically active lectin from Korean mistletoe, Viscum album var. Coloratum

A mistletoe lectin was isolated from water extracts of Korean mistletoe, a subspecies of Viscum album, grown on Quercus mongolica using CM-Sepharose chromatography followed by an affinity chromatography on a concanavalin A-Sepharose column. The compound proved to be a mistletoe lectin II with D-galactose and N-acetyl-D-galactosamine specificity. Matrix-assisted laser desorption time-of-flight mass spectroscopy showed it to have an average molecular mass of 62.7 kDa and to consist of two subunits of 30.6 kDa and 32.5 kDa. It was a basic protein with isoelectric points of 9.4 and 9.6 by capillary isoelectric focusing and was cytotoxic to Molt4 cell. Received 17 November 1998; received after revision 3 March 1999; accepted 3 March 1999     

Some properties of the goat placental lactogen

Summary Goat placental lactogen was partially purified from a medium collected after placental tissue incubation. The data obtained by disc electrophoresis and isoelectric focusing experiments, as well as by means of radioreceptor assay methods, provide evidence of the similarity between the goat and ovine placental lactogen.The careful technical assistance of L. Tichovská is gratefuly acknowledged.     

A rapid assay for the localization of protein kinase modulator (inhibitor) in complete polyacrylamide gels

Summary A gel assay system for determination of inhibitory proteins for protein kinase(s) on isoelectric focusing gels has been developed. Preparations of heat-stable inhibitors were applied, focused, and the complete gels incubated with protein kinase in the presence of substrate protein. At the position where inhibitory protein had focused, the phosphorylation reaction was blocked selectively.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Biochemistry of frog ribonucleases

Frogs have unique pyrimidine base-specific RNases, with structures similar to those of turtle, iguana and chicken RNases. Among the four frog RNases discussed here, three from Rana pipiens, R. catesbeiana and R. japonica oocyte cells show anti-tumour activity, and the latter two show lectin activity towards sialic acid-rich glycoproteins. In this review, (i) we compare their unique primary structures with respect to the locations of their disulphide bridges, three-dimensional structure, base specificity and heat stability as compared with RNase A, and (ii) we summarize current knowledge about the mode of action of lectin and the antitumour activities of the three frog RNases.     

姜黄素逆转人结肠癌细胞HCT-8／VCR的双向凝胶电泳技术的建立及优化

目的建立并优化姜黄素逆转人结肠癌细胞HCT-8／VCR蛋白质纽的双向电泳技术。方法姜黄素作用于人结肠癌细胞HCT-8／VCR,提取蛋白质。对双向凝胶电泳中蛋白质样品的处理,蛋白上样量,IPG胶条的选择,以及聚焦条件等进行调整和每化。结果采用瓶内刮取的方法裂解细胞,pH3-10NL的胶条,上样量200ug,延长除盐时间,聚焦60000伏小时,可以得到分辨率较高、重复性较好的双向电泳图谱。结论初步建立了分辨率较高且重复性较好的姜黄素逆转人结肠癌细胞HCT-8／VCR蛋白质双向凝胶电泳图谱。     

Isoelectric focusing of mosquito esterases in the presence of Triton X-100

Summary Triton X-100 improves nonspecific esterase solubilization from mosquito samples and also leads to increased resolution in an isoelectric focusing electrophoresis.Acknowledgments. This research was supported by a US Army contract/grant DAMD 17-77-C-7018, US Army Medical Research and Development Command, Washington, DC and by the Office of Naval Research.     

Isoelectrically focused carboxyesterases as a biological marker in chimeras

Summary Species-specific multiple forms of carboxyesterases (CE) were determined in zymograms obtained by isoelectric focusing (IEF) using homogenized wing zeugopodal tissues of chick, quail and quail-chick chimeras. The validity of the CE pattern of chimeric tissues was verified by the nuclear marker technique. Analytical IEF of CE was found to be useful for investigation of the origin of tissues in chimeras.Acknowledgment. This work was supported by grant AM21026.     

High resolution isoelectric focusing of juvenile hormone esterase activity from the hemolymph of Trichoplusia ni (Hübner)

Juvenile hormone esterase (JHE) activity from the hemolymph of larval Trichoplusia ni was analyzed by two different isoelectric focusing (IEF) methodologies. Use of techniques capable of progressively higher resolution split ultimately what appeared at lower resolution to be a single peak into two discrete peaks of JHE activity (pI 5.5 and 5.3). Neither peak was a degradation artifact of the other caused by conditions of IEF.     

Soluble proteins of chemical communication in the social wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis>

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.Received 14 May 2003; received after revision 8 June 2003; accepted 12 June 2003     

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys₆-Cys₂₁, Cys₁₃-Cys₃₀, Cys₂₀-Cys₄₈, Cys₃₂-Cys₄₆). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail. Received 23 July 2001; accepted 31 July 2001     

A simple diagnostic method for the differentiation of Tamm-Horsfall glycoproteins from healthy probands and those from recurrent calcium oxalate renal stone formers

Tamm-Horsfall glycoproteins (THPs)^* from healthy probands, and those from a majority of recurrent calcium oxalate renal stone formers, reveal different properties when analyzed using isoelectric focusing. The pl-values of THPs from healthy probands are approximately 3.5 while THPs from recurrent renal stone formers have pl-values between 4.5 and 6. The two groups of THPs exhibit completely different protein patterns in IEF. This proves the structural difference of these THPs. The differences in IEF analysis allow the differentiation between THPs from healthy probands and those from recurrent calcium oxalate stone formers. These differences could possibly by used as a simple diagnostic method for the recognition of recurrent calcium oxalate renal stone formers.     

Large scale isoelectric focusing analysis of the non-histone proteins of the nucleus: Isolation of components with alkaline isoelectric points

Summary After large scale isoelectric focusing of rat liver non-histone protein in polyacrylamide gel, pH range 4–8.6, the only protein material found outside the gradient was present in the cathode solution (20 mM NaOH). This was low mol. wt protein material (approximately 10,000) with an acidic amino acid composition. It bound 5–6 times its own weight of basic ampholine carrier ampholytes to give a complex with a pI of 8.82. This could be dissociated by dialysis against 1 M NaCl.We are indebted to the Yorkshire Cancer Research Campaign for financial support for this project.