Electrophoretic variation of seed proteins among U.S. populations of Tripsacum dactyloides Var. dactyloides

Accessions from across the range of Tripsacum dectyloides in the US. were assayed electrophoretically for interpopulation variation in seed proteins. Clustering of isoelectric focusing patterns for Tris-HCl and water-soluble proteins revealed high levels of homogeneity. Preliminary resultswith alcohol-soluble proteins, in contrast, showed this fraction to be intrinsically much more variable. Divergent functional roles of the protein fractions themselves could account for this observed difference in variation and are discussed in the paper.     

Flavonoid patterns and systematics in Eleusine

A survey of leaf flavonoids was conducted on Eleusine coracana ssp. coracana and ssp. africana, E. indica, E. multiflora, E. tristachya, E. floccifolia, and E. compressa. Twenty phenolic compounds were detected. Those identified were: orientin, isoorientin, vitexin, isovitexin, saponarin, violanthin, lucenin-1, and tricin. The study revealed a general generic flavonoid pattern except for E. compressa, which occupies an isolated position in Eleusine. Flavonoids of the perennial E. floccifolia and the annuals E. multiflora and E. tristachya are markedly different from those of cultivated E. coracana, suggesting that these species are only distantly related to the crop. The morphologically well defined E. coracana—africana—indica group also forms a unit in respect of flavonoids. Subspecies africana exhibits a higher flavonoid similarity to ssp. coracana (finger millet) than does E. indica. The weedy race of ssp. africana usually combines flavonoids of both the wild and domesticated subspecies. The flavonoid pattern of the dedza race of ssp.africana is identical to that of finger millet, suggesting either a direct origin of the crop from this race, or extensive introgression from the crop into ssp. africana. A lack of qualitative differences in flavonoids between cultivated races of finger millet is indicative of the genetic stability of these compounds. The flavonoid data confirms the domestication of finger millet from ssp. africana.     

The sensitivity of isoelectric focusing and electrophoresis in the detection of sequence differences in proteins

Fourteen myoglobins of known sequence were examined by isoelectric focusing with and without urea. The 14 sequences formed six distinct mobility classes on gels without urea and three classes on those with urea. For these proteins, isoelectric focusing provides no advantage over single, nonequilibrium, nondenaturing gels in the total number of distinguishable mobility classes. Only major charge differences, resulting from the changes in the total numbers of acidic and basic amino acids, can be detected on gels with urea, indicating that denaturation by urea alters proteins so that small differences in ionization are eliminated.We thank the Department of Biology, University of Utah, for financial support.     

Immunological relationships and a genetic interpretation of major and minor acidic proteins in human parotid saliva

Isoelectric focusing was performed on parotid salivas selected for their electrophoretic phenotypes of proline-rich acidic salivary proteins. Fractions encompassing narrow pH regions were pooled and examined by polyacrylamide gel electrophoresis. Isoelectric focusing yielded partial purification of major and minor acidic proline-rich proteins which were subsequently compared by immunoelectrophoresis and double immunodiffusion against goat anti-human parotid saliva. Cross-reactivity without spurring between all fractions containing major Pr proteins in both immunoelectrophoresis and double immunodiffusion suggests that these proteins are immunologically very similar or identical.This study was supported in part by an award from the American Cancer Society Institutional Grant IN-88F to Fels Research Institute.     

Properties of albumins of milled rice

Extraction studies on IR36 milled rice showed that albumins solubilized by 0.1–0.15 M (NH₄)₂SO₄ consisted of about 20% high(～5%) lysine, fast-migrating proteins on electrophoresis at pH 8.3 and about 80% lower ～2%) lysine proteins of slower mobility. The 2%-lysine albumins were insoluble in 1.8 M (NH₄)₂SO₄ while the higher lysine albumins required 4 M (NH₄)₂SO₄ to precipitate. The 2%-lysine albumins were not fractionated by gel filtration and gave only one major fraction with MW 19 000. SDS-polyacrylamide gel electrophoresis confirmed the major subunit to be of MW 17 000. These albumins were separated by DEAE-Sephacel chromatography at pH 8.5 into three fractions of similar aminograms but differing in analytical get electrophoretic and isoelectric focusing patterns.     

Fractionation of globulins of milled rice

Globulins were prepared by repeated precipitation with 1.3 M (NH₄)₂SO₄ from a 0.7 M NaCl extract of milled rice. Isoelectric precipitation at pH 4.5 did not effectively remove the α-globulin from the others. A major fraction that remained in solution during dialysis of the globulin precipitate against water was similar in properties to the globulin soluble at pH 4.5 during the isoelectric precipitation process. Some properties of this water-soluble globulin fraction are reported. Proteins extracted from milled rice at 50° with 0.5 M NaCl and precipitated as 1- to 3-μm particles on cooling were verified to be globulins.     

Comparative examination of soluble red muscle proteins of fifteen Sparidae species

Soluble red muscle proteins from the lateral line of 15 Sparidae species were analysed by isoelectric focusing. Species-specific patterns were found. The species-specific protein fractions could be correlated with different metabolic activities and with different growth indexes. More protein fractions were identified for Pagellus acarne (Risso) and Diplodus annularis (L.) than for the other 13 species. This would appear to be related to growth index. The existence of red muscle with a greater number of protein bands than white muscle was confirmed in almost all species. The similarity coefficients have shown that closely related species have similar patterns and, thus, higher similarity coefficients. The derived dendrogram agrees with previous classifications based on morphological information.     

青藏高原东缘常见40种禾本科植物种子大小变异研究

采集青藏高原东缘常见禾本科植物的种子,测量并计算种子的长、宽、高、质量等9项形态学指标,将上述指标分别与海拔高度、种子质量和种子总体方差间进行相关性分析。结果表明：①种子的各表型性状均存在一定程度的变异,其中种子长/高的变异系数最高,而种子质量的变异系数最低,表明种子质量较稳定。②相关性分析表明种子的长与种子质量间有较强的相关性;海拔高度与禾本科种子表型性状之间存在一定程度的相关性,沿海拔梯度,种子长、种子宽、种子高、种子宽/种子长和种子质量逐步减小;总体方差与种子长/种子高具有较高的相关性。认为可以用种子长/高替代总体方差来衡量禾本科种子的整体形态;海拔是影响禾本科种子大小及变异的一个重要因素。     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Study of the soluble white muscle tissue proteins from fifteen Sparidae species

Soluble proteins of white skeletal muscle tissue of 15 species of Sparidae were analysed. Species-specific electrophoretic and isoelectric focusing patterns were found. Some bands exhibited the same mobility at genus level or at subfamily level, others differed significantly.
Considerable similarity was observed in the species of the genera Spams, Pagellus and Diplodus . Significant differences in the protein bands were noted between the contained subfamilies Denticinae, Sparinae and Boopsinae, confirming the existence of three separate phyletic lines within the family Sparidae.
This study has shown that in these species there is a similarity between classifications based on morphological data and those derived from biochemical studies.
Variation within species can be corrected for by carrying out multiple inter-specific comparisons and determining the variance of the similarity coefficients. Closely related species have similar patterns and, thus, higher similarity coefficients.
The discrepancy in similarity matrices based on morphology and white skeletal muscle tissue proteins of sea bream species shows that electrophoretic methods provide additional information relevant to the systematics of fishes.
Further work on comparison on soluble red muscle proteins of these species is proposed.     

Isoelectric focusing of human head hair proteins. Preliminary research

The examination of human hair keratin to obtain genetic information, which may be useful also in forensic sciences, has been carried out with the use of isoelectrophoretic procedure obtaining considerable evidence for the existence of specific-species patterns. In this paper the keratins extracted from hairs of 280 subjects belonging to Sardinian people (113 males, 167 females, aged from 1 to 89, belonging to 52 families) were analyzed using IEF in thin-layer polyacrylamide gel (0.5 mm) in the pH range 2.5–7.0, followed by the silver staining method. Number, position and colour of the bands were the same in all the analyzed samples but a large individual variability was revealed for the relative intensity of some bands. Differences for a long time storage were not revealed as well hair's sample as protein extract: Neither were differences in the number and position of the bands analyzing samples of hair from several sites of the head of the same individual revealed. The results obtained are a useful indication to continue this research considering the numerous fields of application of this analysis system.     

分子系统学研究进展

分子系统学 ( molecular systematics)是近 30年发展起来的一门综合性前沿学科 ,它在分子水平上对生物进行遗传多样性、分类、系统发育和进化等方面的研究 ,其研究结果对于保护生物多样性 (尤其是遗传多样性 ) ,揭示生物进化历程及机理具有十分重要的意义。1　分子系统学的定义及发展简史分子系统学是通过检测生物大分子包含的遗传信息 ,定量描述、分析这些信息在分类、系统发育和进化上的意义 ,从而在分子水平上解释生物的多样性、系统发育及进化规律的一门学科。它以分子生物学、系统学、遗传学、分类学和进化论为理论基础 ,以分子生物学…     

A simple Beckman DU accessory for location of proteins separated by isoelectric focusing in granulated gel layers: Isolation of human serum very low density apolipoproteins C-II and C-III-1

A simple, low-cost gel layer scanner has been developed for the Beckman DU spectrophotometer. The gel scanner makes it possible to localize zones precisely after preparative isoelectric focusing of proteins in 2-mm thick Sephadex G-200 layers. Using the scanner after isoelectric separation of a mixture of human serum apolipoproteins C-III-1 and C-II, pure proteins could easily be obtained.     

Isoelectric focusing following by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region, at least up to pH 10. The best method for developing zymograms and immunoprints appears to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.     

Detection of a new K-casein variant in cow''s milk

In German Simmental cattle a new kappa-casein variant was detected by isoelectric focusing, which cannot be distinguished from kappa-casein A by polyacrylamide and starch gel electrophoresis. It was named kappa-casein D. The frequencies of kappa-casein alleles were kappa-Cn A 0.75, kappa-Cn B 0.24, kappa-Cn D 0.01.     

Low MW gliadin-like proteins from wheat endosperm

A new group of hydrophobic endosperm proteins from Triticum aestivum has been characterized. It consists of 10 components with MWs in the range of 17 000–19 000, which have a similar range of electrophoretic mobilities at pH 3.2 as the classical gliadins. However, they have a higher proportion of sulphur amino acids and lower levels of glutamine and proline than the gliadins.     

Genetic polymorphism of PIF (parotid isoelectric focusing variant) proteins with linkage to the PPP (parotid proline-rich protein) gene complex

Genetic polymorphism is found among the PIF (parotid isoelectric focusing variant) salivary proteins after separation by prolonged isoelectric focusing in pH 3.5–5.2 urea polyacrylamide slab gels subsequently stained for protein. Two PIF proteins are either present (PIF +) or absent (PIF –) from all salivas. The phenotypes are determined by autosomal inheritance of two alleles, PIF ⁺ and PIF ^–. Gene frequencies in randomly collected samples show marked racial differences: among 148 whites, PIF ⁺ is 0.66 and PIF ^– is 0.34; among 90 blacks, PIF ⁺ is 0.35 and PIF ^– is 0.65; among 78 Chinese, PIF ⁺ is 0.56 and PIF ^– is 0.44. Studies in 41 families including 129 children support the interpretation of control of PIF by a single autosomal locus. In 8 PIF+ × PIF– matings, there were 8 PIF– (6.34 expected) children. In 33 PIF+ × PIF+ matings, there were 7 PIF– (6.70 expected) children. Linkage studies indicate that PIF is closely linked to the proline-rich protein (PPP) gene complex (e.g., for six families, lod score at =0.00 of PIF/G1 is 3.58). In 107 randomly collected samples from whites, PIF is strongly associated with Db (x ₁ ² =20.02; P<0.0001) and Gl (x ₁ ² =12.58; P=0.0005) but not with Pr, Ps, Pm, and Pa proteins. These data (probably reflecting genetic disequilibrium) suggest that PIF may be closer to Db and G1 than to other identified loci of the PPP gene complex. The PPP gene complex includes at least seven genes (and probably more) that produce many acidic and basic proline-rich proteins, constituting about two-thirds of parotid salivary proteins that are thought to have important functions at the tooth surfaces.This study was supported by a grant from the National Institutes of Dental Research (DEO 3658-15). Paper No. 2435 of the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706.     

Comprehensive yeast proteome analysis using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-MS/MS

As demonstrated in this study, a CIEF-based multidimensional separation platform not only is compatible with the detergent-based membrane protein preparation protocol, but also achieves both the largest yeast membrane proteome coverage and the most comprehensive analysis of the yeast proteome to date. By using a 1% false discovery rate for total peptide identifications, a total of 2513 distinct yeast proteins are identified from the SDS-solubilized fraction with an average of 5.4 peptides leading to each protein identification. Among proteins identified from the SDS-solubilized fraction, 407 proteins are predicted to contain at least two or more transmembrane domains using TMHMM (www.cbs.dtu.dk/services/TMHMM-2.0/), corresponding to 46% yeast membrane proteome coverage. Only four additional membrane proteins are identified in the soluble and urea-solubilized fractions, affirming the utility of SDS extraction for enriching the membrane proteome. By combining proteome results obtained from the soluble, urea-solubilized, and SDS-solubilized fractions, a single yeast proteome analysis yields the identification of 3632 distinct yeast proteins, corresponding to 55% theoretical yeast proteome coverage or 70% of proteins predicted to be expressed during log-phase growth in rich media.     

Amino acid composition of zein molecular components

Zein extracted from maize endosperm has been fractionated into four polypeptide chains, having the following MWs 23 000, 21 000, 13 500 and 9600. By amino acid analysis the two smaller MW chains (representing 30% of total zeins) have been found to be zein-type molecules. These two chains are thought to be responsible for zein granule formation via -S-S- bridges. Zein is also highly heterogeneous in charge, and is resolved into at least 15 components, with pI's in the pH range 5–9. As demonstrated by amino acid analysis, part of this heterogeneity is due to spot mutations in some of the genes responsible for zein synthesis.     

Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.