Complex Protein Pattern Research Articles

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Rapid differentiation of Artemia spp. Populations by thin layer isoelectric focusing

Artemia spp. of various geographic origins, yearly collection lots, developmental stages, and a binary geographic mixture were analyzed by thin layer polyacrylamide gel isoelectric focusing (IEF). IEF of geographical Artemia samples yielded unique profiles for each population in the pH range employed. Parthenogenetic populations of Artemia exhibited IEF patterns which were, generally, dominated by fewer proteins; bisexual populations exhibited more evenly distributed, complex protein patterns. There were similarities reflected in IEF profiles of geographically isolated Artemia descended from a common ancestry. Yearly collections of Artemia exhibited no apparent differences in IEF protein patterns. There were differences noted in three developmental stages of Artemia. A binary mixture of geographic populations yielded IEF patterns intermediate to those of the component pure samples.

The relationship between polyribosomal and latent membrane-bound messenger ribonucleoprotein particles in artemia embryos

1. 1. Polysomal messenger ribonucleoprotein (mRNP) particles from developing Artemia cysts were isolated, characterized and compared with latent membrane-bound mRNP particles isolated from dormant cysts. 2. 2. The polyribosomal mRNP particles sedimented between 25–35 S in a sucrose gradient and had a buoyant density of 1.33g/cm 3 in Cs 2So 4. 3. 3. Latent particles had a higher sedimentation coefficient and lower buoyant density. 4. 4. The poly(A) + RNA in the two kinds of particles was comparable in size, 10–20S. 5. 5. The protein composition of the particles, as determined by electrophoresis, was different. 6. 6. Polyribosomal particles contained 9 major and 6 minor proteins; a 72 k poly(A)-associated protein was present. 7. 7. Latent particles were characterized by a complex protein pattern ranging in apparent mol. wt between 14,000–140,000. 8. 8. Some proteins with similar molecular weight and isoelectric point were probably common to both kinds of particles.

Plasmodium falciparum: Isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10 9 mature-form parasites, a total of about 10.7 × 10 9 merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.

The production of a membrane by purified oligodendroglia maintained in culture

Purified oligodendroglia maintained in culture produce whorls of membrane lamellae, adjacent to the cell soma. When subcellular fractions are prepared from the cells in culture, three membrane fractions are obtained: a glial light fraction which electron micrographs show to be predominantly large vesicles; an intermediate fraction that on electron micrographs consists of whorls of membrane lamellae; and a plasma membrane fraction consisting primarily of small vesicles. In a study on the production of the membrane lamellae, the results show that there is a rapid incorporation of radiolabel into cerebrosides and phosphatidylcholine, with lower incorporation into other phospholipids. There is a delay in incorporation into sulfatides. Incorporation into proteins show a complex heterogeneous pattern of proteins, ranging from high to low molecular weight (MW) bands. The incorporation data may reflect the composition of the subfractions after different times in culture.

Characterization of a poly(A) +RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A) +RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A) +RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs 2SO 4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17–26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A) +RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A) +RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs 2SO 4-resistant structures, independent of ribosomes, but intimately associated with membrane components.

Double-labeling and high precision comparison of complex protein patterns on two-dimensional polyacrylamide gels

Combination of double-labeling and two-dimensional polyacrylamide gel electrophoresis allows high precision comparison of proteins from two different cultured cell lines. Extracts of cell lines labeled with 14C and 3H, respectively, are mixed and analyzed on a single two-dimensional gel. Fluorography detects both isotopes, but autoradiography through carbon paper detects only 14C. Proteins can be classified into classes common to both cell lines and those specific to each cell line. The method should be useful for a wide range of biochemical studies.

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm 2 of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of 14C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.

Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster.

Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with35S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.

Structure of transcriptionally active chromatin.

Rat-liver chromatin has bee fractionated into transcriptionally active and inactive regions [Gottesfeld et al. (1974) Proc. Nat. Acad. Sci. USA 71, 2193-2197] and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by tne endonuclease DNase II. The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nuclease-resistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition.

Membrane adenosine triphosphatase of Micrococcus lysodeikticus: effect of millimolar Mg2+ in modulating the properties of the membrane-bound enzyme.

The latency of Micrococcus lysodeikticus membrane-bound Mg(2+)-adenosine triphosphatase (ATPase) is expressed by the ratio of its activity assayed in the presence of trypsin ("total") versus the activity assayed in absence of the protease ("basal"). By isolating membranes in the presence of variable concentrations of Mg(2+) (50 mM, 10 mM, or none) and by washing them with different Mg(2+)- and ethylenediaminetetraacetic acid-containing tris(hydroxymethyl)aminomethane-hydrochloride buffers (pH 7.5), we showed that the enzyme latency was dependent on the environmental concentration of this divalent metal ion. Mg(2+) bound to at least two classes of sites. The binding of Mg(2+) to low-affinity sites (saturation at approximately 40 mM external Mg(2+)) induced a high basal ATPase activity, whereas its binding to medium-affinity sites (saturation at about 2 mM Mg(2+)) correlated with low basal activity and a very high stimulation by trypsin. Membranes with tightly bound Mg(2+) (high affinity?) revealed an intermediate behavior for the latency of M. lysodeikticus ATPase. The Mg(2+)/Ca(2+) antagonism as activators of the membrane ATPase was not directly related to Mg(2+) binding by the membranes. The efficiency of the ATPase release from M. lysodeikticus membrane by 3 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) was inversely proportional to the concentration of external and/or bound Mg(2+). Deoxycholate (DOC) (1%) solubilized the ATPase from all types of membrane. All the soluble ATPases behaved as Ca(2+)-ATPases, but the DOC-soluble fractions showed degrees of latency like those of the original membranes. The DOC-soluble ATPase preparation revealed a vesicular structure and complex protein patterns by sodium dodecyl sulfate gel electrophoresis. We propose that ATPase latency is modulated via a Mg(2+)-ATPase-membrane complex.

Molecular organization in bacterial cell membranes III. Components of a “soluble” fraction obtained by n-butanol extraction of Streptomyces albus membranes

We have isolated a “soluble” fraction of Streptomyces albus G membranes or membranes previously solubilised by sodium dodecylsulphate, using n-butanol extraction. Polyacrylamide gel electrophoresis in sodium dodecylsulphate of the whole membrane showed a complex protein pattern (about 20–25 bands) with two predominant groups. The “soluble” fraction represented about 25% of the membrane protein and contained part of the major polypeptides. The yield of protein in “soluble” form decreased when membranes were suspended in water and di not significantly change if membranes were reduced with sodium dithionite and then treated with iodoacetamide. A change in relative mobility of some of these polypeptides seemed to occur with membrane delipidation. The proteins of the fraction appear to be glycoproteins as indicated by their simultaneous staining for protein and carbohydrate and the parallel sensitivity to trypsin of both stains. The apparent molecular weights by sodium dodecylsulphate gel electrophoresis of the proteins (glycoproteins) were: 63 000, 40 000 and 17 000. Similar protein patterns were obtained by extraction of the membranes with EDTA and non-ionic detergents. Lipid and nucleotide material were also found in the “soluble” fraction. The “soluble” fraction showed by gel filtration on Sephadex G-200 the existence of different states of aggregation. These states of aggregation revealed the same electrophoretic pattern of proteins, which seemingly corresponded to that of the original fraction (i.e. three protein groups with relative mobilities 0.65, 0.80 and 1.0). Treatment of the samples under different conditions with 1% dodecylsulphate (supplemented or not with 0.5% β-mercaptoethanol) failed to completely dissociate the fraction as shown by Sephadex filtration.

The ribosomes of Drosophila. I. Subunit and protein composition.

Functional ribosomes from Drosophila virilis dissociate to form 40S and 55S subunits. One-dimensional gel electrophoresis in either dodecyl sulfate-acrylamide gels (Fig. 1) or urea-acrylamide gels (Fig. 2) reveal complex protein patterns. Increased resolution in two-dimensional acrylamide gels (Figs. 3 and 4) indicate the existence of at least 28 proteins in the small subunit, and 39 proteins in the large subunit. Molecular weight estimates indicate a molecular weight range (and average) of 16500–40000 (25800) for the 40S subunit proteins, and 12500–45000 (24300) for the 55S subunit proteins. Comparison of larval and adult ribosomal protein patterns did not reveal qualitative differences between them.

Genetic Variability and Relationships in Pacific Salmon and Related Trout Based on Protein Variations

Utter, F. M., F. W. Allendorf and H. 0. Hodgins (Northwest Fisheries Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Division of Environmental Conservation, 2725 Montlake Boulevard East, Seattle, Washington 98112) 1973. Genetic Variability and Relationships in Pacific Salmon and Related Trout based on Protein Variations. Syst. Zool. 22:257-270.-An investigation was made of the biochemical genetic variation within and among Pacific salmon (Oncorhynchus spp.) and related trout (Salmo spp.). Estimates (based on 19 to 23 loci) of proportion of loci polymorphic and average heterozygosity respectively were: pink salmon (O. gorbuscha)-.111 and .014, chum salmon (O. keta)-.100 and .006, sockeye salmon (O. nerka)-.087 and .018, coho salmon (O. kisutch)-.130 and .018, chinook salmon (O. tshawytscha)-.130 and .015, and rainbow trout (S. gairdneri)-.261 and .037. The differencesof these two parameters between populations of salmon and rainbow trout were significant and may reflect the greater habitat diversity of rainbow trout contrasted with Pacific salmon species. Interspecies comparisons were made among the above species plus masu salmon (O. masou) and cutthroat trout (S. clarkii), based on allelic proteins of eight loci and more complex protein patterns assumed to reflect four additional loci. A dendrogram, constructed from indices of similarity which reflected pairwise interspecies protein differences, separated the species into two major groups; one group contained the two trout species which were paired closely and-more distantly-the masu salmon, while the other group contained the remaining five species of salmon. In the latter group the chinook and coho salmon were paired together as were the pink and sockeye salmon; the chum salmon was intermediate between the two subgroups.

Afrikander cattle congenital goiter: purification and partial identification of the complex iodoprotein pattern.

A genetically determined goiter of Afrikander cattle has been shown not to contain any normal thyroglobulin. Apart from iodinated serum proteins such as albumin and immunoglobulin an unusually complex protein pattern was found. Iodoproteins with sedimentation coefficients of 9S, 12S and 18S were isolated and purified in ultracentrifugally homogeneous forms by gel chromatography and sucrose gradient ultracentrifugation. A 4S component which did not contain any significant 125I label was also purified. The 12S iodoprotein was the only component which formed a precipitin complex with antithyroglobulin antibodies. Its hydrodynamic properties closely resembled those of the 12S half—molecule subunit of thyroglobulin. However, no association of the 12S iodoprotein to thyroglobulin was detected. The interrelationship of the 9S and 18S iodoproteins and their relationship to thyroglobulin was established by their reaction with anti–12S iodoprotein antiserum. The 9S and 12S iodoproteins contained most of the goiter ...

Serum protein variations in horses.

IT is now clear that intraspecific serum protein variation is widespread in mammals at least, and that the technique of starch-gel electrophoresis developed by Smithies1 is a powerful tool for revealing these differences. Complex protein patterns have been obtained with sera from horses, due to concomitant variation in several protein systems.