Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca²⁺ (in the absence or presence of calmodulin) or independent of these factors. In these present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.     

Two isoforms of 17-kDa essential light chain of aorta media smooth muscle myosin

Aorta myosin contains two kinds of light chain, 20-kDa phosphorylatable light chain and 17-kDa essential light chain (LC17). Purified myosin from porcine aorta media showed 3 distinct light chain bands on polyacrylamide gel electrophoresis (PAGE) in the presence of urea (urea-PAGE). The mobilities of the faster two components did not change after incubation of the myosin with a myosin light chain kinase. Gel slices containing the faster two bands were separately subjected to PAGE in the presence of sodium dodecylsulfate. Both components showed identical mobility with that of LC17. The two components were designated as LC17a and LC17b in increasing order of mobility on urea-PAGE. They were isolated by DEAE-Toyopearl ion exchange column chromatography. The amino acid compositions of LC17a and LC17b were similar to each other, but the contents of Ser, Met, Ile, and His were distinctly different. These results suggest that the two components are isoforms. The ratio of the content of each isoform (LC17a: LC17b) in the purified porcine aorta myosin was 39:61, and essentially the same ratio was found with washed muscle homogenate of porcine aorta. Then washed aorta muscle homogenates of rabbit and rat were examined. Two bands having similar mobilities to those of porcine homogenate were also found in urea-PAGE. The ratios of the two components were 31:69 and 66:34, respectively, for rabbit and rat. Aorta smooth muscle thus may contain many types of isomyosin.     

Subcellular distribution and properties of guanylate cyclase in rat cerebellum.

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.     

Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.     

Affinity purification of cytosolic epoxide hydrolase from human, rhesus monkey, baboon, rabbit, rat and mouse liver

An affinity purification system based on elution of cytosolic epoxide hydrolase from a methoxycitronellyl thiol ligand with 4-azidochalcone oxide was applied to a variety of samples including liver from human, monkey, baboon, rabbit, rat and mouse as well as mammary gland from mouse. Hepatic tissues yielded a major 58 kDa band on SDS-PAGE, but the system had to be modified slightly to remove a 33 kDa band for rat. All of the affinity purified hydrolases showed similar properties with regard to substrate selectivity, pH dependence and mobilities on SDS-PAGE.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degrees C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent pattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (P. Backlund, pers. comm.). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designated as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 microg of protein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relative mobility (Rf). Two or three gel slices, corresponding in Rf to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cross-linked) of 18 mm diameter, equipped with a collection chamber of 200 microL volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.     

Rate constants studies of the dye-sensitized photoinactivation of lysozyme

Summary The rate constants for the photodynamic inactivation of hen egg-white lysozyme at different temperatures were studied. Arrhenius plots of the methylene blue sensitized photo-inactivation of lysozyme gave an experimental activation energy of 7.5 kcal/mol. The rate constants for the photodynamic inactivation of lysozyme in the presence of riboflavin decreased almost linearly in the temperature range 4–38° C. The photosensitized oxidation of lysozyme at –20° C in freezing and non-freezing solvents was possible only in the presence of riboflavin. The effect of dye concentration on the quantum yield and rate constant for the photodynamic inactivation of lysozyme was examined. The quantum yields were lower when the concentrations of methylene blue used were low, and increased on increasing dye concentration, getting to a maximum and then declined at higher dye concentrations. It was found that in the case of riboflavin sensitized photo-inactivation of lysozyme both the rate constant and the quantum yield increased as the dye concentration increased. No maximum was observed over the range of dye concentrations studied. A new mechanism is postulated for the photodynamic action of lysozyme in the presence of riboflavin.     

Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat. General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca2+ (in the absence of presence of calmodulin) or independent of these factors. In the present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.     

Decolorization of azo dyes and simulated dye bath wastewater using acclimatized microbial consortium--biostimulation and halo tolerance

Anaerobic acclimatization of activated sludge from a textile effluent treatment plant to high concentration of RB5 could effectively decolorize RB5 dye solution. The strains viz. Pseudomonas aeruginosa and Bacillus circulans and other unidentified laboratory isolates (NAD1 and NAD6) were predominantly present in the microbial consortium. The conditions for efficient decolorization, biostimulation to increase effectiveness of microbial consortium, its tolerance to high salt concentration and non-specific ability towards decolorization of eight azo dyes, are reported. The optimum inoculums concentration for maximum decolorization were found to be 1-5 ml of 1800+/-50 mg l(-1) MLSS and 37 degrees C, respectively. The decolorization efficiency was 70-90% during 48 h. The biomass showed efficient decolorization even in the presence of 10% NaCl, as tested with RB5. In the presence of flavin adenine dinucleotide (FAD) more than 99% decolorization occurred in 8h. The decolorization of RB5 was traced to extracellular enzymes. The effectiveness of acclimatized biomass under optimized conditions towards decolorization of two types of synthetic dye bath wastewaters that were prepared using chosen azo dyes is reported.     

Immunological and biochemical properties of metallothioneins of golden hamster,mouse and rat

Summary Golden hamster, mouse and rat hepatic cadmium metallothioneins (MT) were purified by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and activated Thiol-Sepharose 4B affinity chromatography. Metallothioneins were separated by DEAE-Sephadex A-25 chromatography into two forms: MT-1 and MT-2. In mouse and golden hamster liver, MT-1 was the major form. The purified proteins were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In non-denaturing polyacrylamide gel electrophoresis, migration of mouse, rat and golden hamster hepatic metallothioneins were found to be different. Antibodies to mouse hepatic MT-1 was raised in rabbits. The antiserum cross reacted with mouse and hamster MT-1 and MT-2 giving a single precipitin band. Mouse, rat and hamster hepatic MTs are immunologically identical but electrophoretically different. The kidney and pancreatic MTs of rat and golden hamster were purified by Sephadex G-75 gel filtration. They were immunologically distinct. Pancreas MT formed a line of partial identity with hepatic MTs. Kidney MTs form two precipitin band one identical with the pancreatic form and another of complete identity with the hepatic MTs. This indicates the presence of tissue specific MTs.     

The effect of donor age on the sensitivity of osteoblasts to the proliferative effects of TGF(beta) and 1,25(OH(2)) vitamin D(3)

The loss of osteoblast function in aging bone is one of the major causes of osteopenia, or loss of bone mass. In this study, this loss of function was investigated by examining the proliferative response of rat long bone periosteal osteoblasts to TGF(beta1) and 1,25-dihydroxy vitamin D(3) (1,25-D(3)) as a function of donor age. Using a DNA binding fluorescent dye, DNA levels were measured in osteoblast cultures derived from either young adult (3-4 months) or old (14-15 months) rats following treatment with two concentrations (10(-9) M or 10(-12) M) of either 1,25-D(3) or TGF(beta1) or with vehicle. Cells from young rat bone, when treated with 1, 25-D(3), showed a dose-dependent increase in proliferation when treated with the higher dose and a decrease in proliferation when treated with the lower dose. Osteoblasts isolated from old rats did not respond to 1, 25-D(3) treatment. A similar pattern of response to TGF(beta1) was found. When treated with 10(-9) M TGF(beta1), the rate of proliferation increased for young rat osteoblasts, but the old rat derived cells were unresponsive. The 10(-12) M dose of TGF(beta1) was ineffective for both young and old cells. This study has shown that osteoblasts derived from old donors are impaired in their ability to respond to vitamin D and TGF(beta), two of the major controlling factors of skeletal development and maintenance.     

Fluorescence spectra of cells stained with a DNA-specific dye, measured by flow cytometry

Fluorescence spectra of ethanol-fixed rat thymocytes stained with the DNA-specific dye Hoechst 33258 have been measured in an arc lamp-based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5-10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA-specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.     

C-terminal phosphorylation of murine testis-specific histone H1t in elongating spermatids

Previous studies gave differing results as to whether the testis-specific histone H1t was phosphorylated during rodent spermatogenesis. We show here that histones extracted from germ cell populations enriched with spermatids at different stages of development in rat testes reveal an electrophoretic shift in the position of H1t to slower mobilities in elongating spermatids as compared to that from preceding stages. Alkaline phosphatase treatment and radioactive labeling with (32)P demonstrated that the electrophoretic shift is due to phosphorylation. Mass spectrometric analysis of histone H1t purified from sexually mature mice and rat testes confirmed the occurrence of singly, doubly, and triply phosphorylated species, with phosphorylation sites predominantly found at the C-terminal end of the molecule. Furthermore, using collision-activated dissociation (CAD) and electron transfer dissociation (ETD), we have been able to identify the major phosphorylation sites. These include a new, previously unidentified putative H1t-specific cdc2 phosphorylation site in linker histones. The presence of phosphorylation at the C-terminal end of H1t and the timing of its appearance suggest that this post-translational modification is involved in the reduction of H1t binding strength to DNA. It is proposed that this could participate in the opening of the chromatin fiber in preparation for histone displacement by transition proteins in the next phase of spermiogenesis.     

Effect of the Ca ionophore A-23187 on the plasmatic and mitochondrial potentials of the brain synaptosomes in rats: fluorescence measurements

The applicability of the potential-sensitive dye diS-C3-(5) for the study of A23187 + Ca2+ induced plasma membrane hyperpolarization was tested in rat brain synaptosomes. An appropriate dye synaptosome ratio was chosen for the fluorescence titration dye in Ca-free Krebs-Ringer solution. The fluorescence intensity of the probe was increased upon the addition of Ca2+ (1 microM) to the synaptosomes in the presence of A23187 (1 microM). The effect of Ca2+ + A23187 persisted in a Na+-free medium or when Na+ channels were inhibited by tetrodotoxin as well as in high K+-depolarized synaptosomes (75 microM KCl). In the presence of oligomycin or a protonophore (1 microM) the effect of Ca2+ + A23187 was suppressed. This suggests that the A23187-induced fluorescence increase is due to a depolarization of intrasynaptosomal mitochondria. Therefore, the use of the dye diS-C3-(5) for the study of Ca-induced hyperpolarization does not seem to be feasible unless a quantitative model of changes in fluorescence related to the plasma and mitochondrial membrane potentials is elaborated.     

Characterization of the mRNA''s for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.     

Glutathione conjugation by isolated lung cells and the isolated, perfused lung. Effect of extracellular glutathione

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1-chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of 1-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH.     

Tissue specificity of tropomyosin from the crayfish, Cambarus clarki

The molecular heterogeneity and tissue specificity of crustacean tropomyosin were investigated, using muscle and nonmuscle tissues from the crayfish, Cambarus clarki. In muscle, three types of tropomyosin isoforms were found on two-dimensional gel electrophoresis. One of them was specific to cardiac muscle, and the other two were shared by skeletal and visceral muscles. In nonmuscle tissues, four types of isoforms were found on two-dimensional gel electrophoresis and in immunoreplica tests using an antiserum against crayfish skeletal muscle tropomyosin. Two of them were common to the muscle isoforms, but the other two were not detected in muscles. Furthermore, nonmuscle tissues contained several peculiar isoforms, the electrophoretic mobilities of which were considerably higher than those of the other isoforms mentioned above. When tropomyosin was purified from the mid-gut gland, these isoforms with high mobilities were found in the crude tropomyosin preparation. These results showed that the crayfish tropomyosin was heterogeneous and that the isoforms were distributed in a tissue-specific manner, like vertebrate tropomyosin. However, the results did not coincide with those of our previous study on horseshoe crab tropomyosin, which showed molecular heterogeneity but no tissue specificity. In view of the difference in the isoform distributions between the two major groups (Crustacea and Merostomata) of Arthropoda, the significance of the tissue specificity of tropomyosin isoforms was discussed.     

Biodegradation of benzidine based azodyes Direct red and Direct blue by the immobilized cells of Pseudomonas fluorescens D41

Benzidine based azodyes are proven carcinogens, mutagens and have been linked to bladder cancer of human beings and laboratory animals. The textile and dyestuff manufacturing industry are the two major sources that released azodyes in their effluents. The dye, Direct blue contains two carcinogenic compounds namely benzidine (BZ), 4-amino biphenyl (4-ABP), while the dye Direct red has benzidine (BZ). Among 40 isolates of Pseudomonas fluorescens screened, one isolate designated as D41 was found to be capable of extensively degrading the dyes Direct blue and Direct red. Immobilized cells of P. fluorescens D41 efficiently degraded Direct red (82%) and Direct blue (71%) in the presence of glucose.