Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle

One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis. Electrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to be: 2A less than 2X less than 2B less than beta/slow.     

Electrophoretic and immunological characterization of pollen protein ofZea mays races

Electrophoretic and serological studies with proteins extracted from the pollen of 12 races of maize (Zea mays) have proved valuable in distinguishing among these races. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double immunodffusion (Ouchterlony method) were used to analyze the extracted pollen proteins. Jaccard's similarity coefficient was calculated from the data generated by 2-state scoring of the band patterns obtained in each system. Average linkage cluster analysis of the similarity matrices was used to construct phenograms illustrating the similarity among the extracts. The racial groupings indicated in these pheno grams, when compared with those of several morphological studies, show some general agreement. However, there are differences, the most notable being that Chalqueno appeared the most dissimilar of the races studied and that Dulce showed high similarity to Palomero Toluqueño, rather than being an exceptional race as it is usually considered. The possible bearing of the groupings of the races in these phenograms with respect to genetic lineage and selection of maize is discussed.     

Localization of neurophysin in the rat supraoptic nucleus

Summary Localization of neurophysin in neurons of the supraoptic nucleus was accomplished using an unlabeled-antibody, post-embedding, immunoperoxidase technique. Neurophysin was exclusively associated with neurosecretory granules within cell bodies of supraoptic neurons and their processes.Supported by U.S.P.H.S. Grant HD-08867     

Phylogenetic cross-reactivities of monoclonal antibodies produced against rat neurophysin

Previously described mouse monoclonal antibodies against rat neurophysins [Ben-Barak, Y., et al. (1985); Whitnall, M. H., et al. (1985)] were studied here for their cross-reactivities to neurophysins (NPs) from other vertebrate species. Posterior pituitary extracts from various mammals (rat, mouse, cow, human) and lower vertebrates (frog, ratfish) were studied. The monoclonal antibodies displayed several distinct patterns of cross-reactivity to the various species, indicating that the epitopes which they recognized were different. PS 67 bound strongly to rat pituitary extract in solid-phase radioimmunoassay (RIA) but showed no cross-reactivity with extracts from any of the other species tested, including the mouse. PS 36 cross-reacted with mouse and frog extracts but showed almost no cross-reactivity with cow and none to ratfish extracts. PS 41 cross-reacted with mouse, cow, and frog extracts. PS 45 was the most cross-reactive antibody and recognized an antigen in extracts from mouse, cow, frog, and ratfish pituitaries. Electrophoresis of proteins extracted from posterior pituitaries, followed by immunoblot staining with either PS 36 or PS 45, demonstrated that the NP-like molecules within each species are heterogeneous, i.e., more than two bands stained in each species. The frog NP stained by PS 45 was about twice the molecular weight of the mammalian NPs. The possible valve of the PS 45 antibody for future molecular cloning experiments on the arginine vasotocin precursor in lower vertebrates is discussed.     

Electrophoretic and immunological study of myosin light chains from freshwater teleost fishes

White muscle myosin light chains from nine freshwater teleosts show a qualitative and quantitative variability on PAGE without phylogenetic correlation. They look different from their higher vertebrate counterparts mainly with regard to electric charge and relative amounts of alkali light chains corresponding to various contents of isoenzymic forms of white muscle myosin. Antibodies against carp white myosin LC1 recognize almost entirely white muscle LC1 from the other fishes and to a lesser degree LC1 from other muscles and vertebrates. The primary structure of this light chain is thus relatively constant. LC2 from carp cardiac muscle and mammalian slow and cardiac muscle do not react at all.     

Polymorphism of alpha-actinin. Electrophoretic and immunological studies of rabbit skeletal muscle alpha-actinins

Heterogeneity of alpha-actinins from rabbit skeletal muscles was studied. Polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate has made it possible to distinguish two closely related alpha-actinins from rabbit fast, white muscle. One isoprotein (designated type I alpha-actinin) appears to be preferentially located in the psoas muscle, while the other (designated type II alpha-actinin) appears to be preferentially located in the longissimus dorsi muscle. Electrophoretic analyses have further shown that the two isoproteins are present as mixtures in most rabbit white, fast-twitch muscles. A standard polyacrylamide gel--sodium dodecyl sulfate/polyacrylamide gel sequential electrophoretic procedure was developed to resolve the different alpha-actinin dimers and to determine their subunit compositions. By this technique, both type I and type II alpha-actinins appeared to be homodimers. No heterodimeric species of alpha-actinin were detected. alpha-Actinin of red, slow-twitch muscles was similar to type II alpha-actinin of fast, white muscle on one-dimensional and two dimensional gels. However, slow, red muscle alpha-actinin was significantly different from fast, white muscle alpha-actinins in terms of one-dimensional peptide mapping and immunological cross-reactivity.     

Electrophoretic and immunological analysis of lipopolysaccharides of Xanthomonas albilineans from three geographical regions

Lipopolysaccharides (LPS) were extracted from the type species of the sugarcane leaf scald pathogen Xanthomonas albilineans from Fiji and additional isolates from Australia, Mauritius and South Africa. After resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the LPS were visualized using a modified silver staining method and analysed by densitometric scanning. Three distinct electrophoretic patterns were identified: the LPS pattern of the Fijian isolate differed from those of the other isolates. Immunological cross-reactivity of the LPS and gamma globulins was assessed by immunoblotting. Immunoblotting showed that although IgG raised against other isolates cross-reacted with the LPS of one another, they did not react with the purified LPS of the Fijian isolate. Differences in LPS patterns and serological cross-reactivity may be due to differences in the length and conformation of the O-polysaccharide chains of the LPS.     

Electrophoretic and immunological characteristics of non-histone proteins of human skin tumors. I) Methods

The object of the research is to classify the nuclear proteins of the cellular nucleus of some types of human skin tumours (melanotic and amelanotic melanomas, squamous epitheliomas). Nuclear proteins were extracted from the nuclei with a saline buffer. Nuclear proteins were extracted from a melanoma step by step. After prolonged centrifugation to sediment the DNA and the large ribonucleoprotein complexes, the nuclear proteins were chromatographed on columns of Biorex 70+ to remove proteins were chromatographed on columns of Biorex 70+ to remove the histones. Nuclear proteins from a squamous epithelioma were injected into a rabbit to induce specific antibodies.     

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis

32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.     

Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

Summary Revertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.Paper No. 60 on Ribosomal Proteins. Preceding paper is by B. Wittmann-Liebold, Hoppe-Seyler's Z. physiol. Chemie, in press.     

Electrophoretic polyamine transport in rat liver mitochondria

Summary Naturally occurring polyamines (spermidine, putrescine, cadaverine), as the well studied spermine, are transported into rat liver mitochondrial matrix provided that mitochondria are energized and the electrical membrane potential has a value of about 180 mV. This condition is achieved by the presence of inorganic phosphate, or acetate, or nigericin in the incubation medium. Valinomycin plus K⁺ almost completely blocks polyamine transport.The obtained results clearly show that all naturally occurring polyamines are transported by an electrophoretic mechanism in responce to a high negative inner electrical potential.The distribution ratio of polyamines across the mitochondrial membrane is far from the thermodynamic equilibrium by many orders of magnitude. This result might suggest the existence of a different pathway for polyamine efflux.     

Electrophoretic protein profiles of rat gonads during differentiation

The protein patterns of fetal rat gonads (14 1/2-21 1/2 days of gestation) were examined by SDS gel electrophoresis. Male gonads contained more protein components at all stages.     

Electron microscope immunohistochemical localization of neurophysin in the rat with hereditary diabetes insipidus

In order to study the subcellular distribution of neurophysin in the rat with hypothalamic hereditary diabetes insipidus (DI), an immunoelectron microscopic localization of neurophysin was performed in the hypothalamo-neurohypophysial system of both homozygous and heterozygous DI rats. Whereas in control rats neurophysin was localized in the granules present in the secretory neurons, in the homozygous DI rats neurophysin was found in the granules and outside the granules in the perikarya and axons of neurons of both supraoptic and paraventricular nuclei. In the heterozygous DI rats findings similar to those observed in homozygous DI rats were observed, although in the posterior pituitary, the exgranular material appeared to be less abundant than in homozygous DI rats. These results clearly demonstrated that in hyperstimulated neurons neurophysin was distributed in both granular and extragranular compartments.     

Dogfish metallothionein--II. Electrophoretic studies and comparison with rat metallothionein

A low-molecular weight metal-binding protein has been isolated and characterized as metallothionein in the liver of the dogfish (Scyliorhinus canicula). This protein is present in both male and female animals and both control and cadmium-treated (both water and i.p. administration) animals as revealed by SDS-PAGE studies. Dogfish metallothionein shows the same metal-dependent anomalous behaviour as rat metallothionein in SDS-PAGE. SDS-PAGE is a good method to identify a protein as metallothionein.