Tubulin and high molecular weight microtubule-associated proteins as endogenous substrates for protein carboxymethyltransferase in brain

The endogenous substrate for protein carboxymethyltransferase in brain was examined. Several polypeptides were methylated when brain slices were incubated with L-methionine or when subcellular fractions of brain, such as the cytosolic fraction, were incubated with S-adenosyl L-methionine. Two methyl-accepting proteins in the cytoplasm were identified as tubulin and high molecular weight microtubule-associated proteins (300 kDa), which are components of microtubules. Tubulin behaved as a 43 kDa protein in acidic polyacrylamide gel electrophoresis, but as a 55 kDa protein in SDS-polyacrylamide gel electrophoresis. The methyl moiety transferred to these proteins from L-methionine was labile at alkaline pH. The high molecular weight microtubule-associated proteins showed higher methyl-accepting activity than tubulin or ovalbumin, which was used as a standard substrate: about 20 mmol of high molecular weight microtubule-associated proteins, 2 mmol of tubulin and 10 mmol of ovalbumin were methylated per mol of each protein in 30 min under the experimental conditions used.     

Colchicine inhibition of plasma protein release from rat hepatocytes

Colchicine, both in vitro and in vivo, inhibits secretion of albumin and other plasma proteins. In vitro, secretion by rat liver slices is inhibited at 10-minus 6 M with maximal effect at 10-minus 5 M. Inhibition of secretion is accompanied by a concomitant retention of nonsecreted proteins within the slices. Colchicine does not inhibit protein synthesis at these concentrations. Vinblastine also inhibits plasma protein secretion but lumicolchicine, griseofulvin, and cytochalasin B do not. Colchicine also acts in vivo at 10-25 mumol/100 g body weight. Inhibition of secretion is not due to changes in the intracellular nucleotide phosphate levels. Colchicine, administered intravenously, acts within 2 min and its inhibitory effect lasts for at least 3 h. Colchicine has no effect on transport of secretory proteins in the rough or smooth endoplasmic reticulum but it causes these proteins to accumulate in Golgi-derived secretory vesicles.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Purification and characterization of ethylene inducing proteins from cellulysin

Ethylene inducing proteins were partially purified and characterized from the cell wall digesting enzyme mixture, Cellulysin. Purification included binding to Sephacryl S-200, isoelectric focusing, molecular sieving on Sephadex G-75, agarose electrophoresis, and sizing using a Superose 12 column. At least three active proteins were obtained from the Sephadex G-75 fraction that move towards the cathode during nondenaturing agarose electrophoresis. These three protein fractions separated by preparative agarose electrophoresis contain polypeptide patterns that are very similar on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions contain three main Coomassie blue stained bands of about 10, 14, and 18 kilodaltons. Gel filtration of the major fraction on a Superose 12 column yields an active peak with an apparent molecular weight of 27,000. Proteolytic enzymes, in the presence of urea, destroy the ethylene inducing activity. We conclude that the ethylene inducing factor (EIF) that we have isolated from Cellulysin is protein. Similar ethylene inducing factors are present in Cellulase RS. Ethylene inducing components from pectinase, Pectolyase, and Rhozyme do not bind to Sephacryl like EIF from Cellulysin. Thus, the components responsible for the ethylene inducing activity in these latter enzyme preparations differ from that of EIF.     

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.     

A Remeasurement of the Molecular Weights of T-Even Bacteriophage Substructural Proteins

T-even bacteriophage substructural proteins were studied by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that tail fibers are composed of two major proteins of 155,000 and 120,000 daltons molecular weight and four minor proteins of 51,000, 38,000, 27,000, and 23,000 daltons. Tail tubes were composed of one predominant protein of 18,500 daltons and one minor protein of 35,000 daltons molecular weight. Tubular polyheads obtained from a T4D amber mutant and by treatment of T4B-infected cells with L-canavanine were also examined, and no significant differences were noted in the molecular weight of the P23 protein.     

Protein synthesis by astrocytes in primary cultures

Protein synthesis, measured as leucine incorporation into acid-precipitable proteins, was determined in astrocytes in primary cultures obtained from the cerebral hemispheres of newborn mice. As can be expected for eucaryotic, ribosomal protein synthesis, the incorporation was almost completely inhibited by cycloheximide (0.01 mM), but unaffected by chloramphenicol (0.03 mM). The rate of synthesis, measured during exposure to a high (0.8 mM) concentration of leucine was 5.4 nmol/hr/mg protein in mature (i.e., at least 4-week-old) cultures. This value is at least twice as high as the protein synthesis rates reported for the adult brain in vivo, suggesting that a very considerable part of the protein synthesis in the adult brain may take place in astrocytes. The molecular weight distribution of the synthesized proteins was determined by polyacrylamide gel electrophoresis, demonstrating synthesis of at least 50 different polypeptides, ranging in molecular weight between 190,000 and 27,000 daltons. The pattern of the synthesized proteins underwent considerable alteration with age in young cultures in which the total content of protein was still increasing, but it was remarkably stable after the age of two weeks. Exposure to dibutyryl cyclic AMP, which is known to alter morphology, content of glial fibrillary acidic protein (GFA), and activities of certain enzymes in the cultured astrocytes, caused marked alterations in the pattern of the synthesized proteins.     

Differences of proteins in isolated egg surface after fertilization of Xenopus laevis

Egg surface proteins of Xenopus laevis were compared between unfertilized and fertilized egg surfaces before the first cleavage. The egg surfaces were isolated in acetone. The macromolecular compositions of egg surfaces were analyzed by two-dimensional gel electrophoresis and were shown to contain at least 30 proteins with molecular weights ranging from 27,000 to 200,000. At 50 min after fertilization, one spot with a molecular weight of 160,000 disappeared and two bands with molecular weights of 190,000 and 180,000 increased gradually after fertilization. Although the disappearance of the spot was not affected by colchicine or cytochalasin B, intensification of the two bands was inhibited completely by the two agents.     

Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.     

Prenyltransferase from Gossypium hirsutum

A protein fraction has been purified from Gossypium hirsutum var. Coker 413 which synthesized all four geometrical isomers of farnesyl pyrophosphate from isopentenyl pyrophosphate alone, from isopentenyl pyrophosphate and geranyl or neryl pyrophosphate. Electrophoretic analysis showed that this protein fraction consisted of three proteins. One of these proteins contained isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase activity. The other two proteins were insufficiently pure to characterize. Estimation of molecular weights by electrophoresis of the three proteins revealed values in the order of 3 × 10⁴ to 1.3 × 10⁵. However the same protein fraction eluted as one peak from Sepharose 6B molecular sieve columns, indicative of a larger protein component as could be accounted for by the electrophoretic molecular weight estimation. From these results and from the different products synthesized it is proposed that isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase and prenyltransferase (farnesyl pyrophosphate synthetase) exists as a multiprotein complex in G. hirsutum.     

The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation

1. On incubating cerebral-cortex slices at 37° in an oxygenated medium marked changes resulted in the subcellular distribution of proteins and phosphoproteins in the tissue. The protein content of the nuclear fraction more than doubled, whereas the yields of microsomal and supernatant proteins were both markedly decreased. The amount of phosphoprotein/mg. of protein decreased in the microsomal and supernatant fractions, but showed little change in the nuclear and mitochondrial fractions. The loss of microsomal protein could be partly prevented by rinsing the slices briefly in cold sucrose solution before dispersion; the altered subcellular distribution was apparently related to contamination of the dispersing solution with traces of salts from the medium. 2. The subcellular location of the phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro has been reinvestigated by two different procedures. Comparison between unstimulated and stimulated slices after incubation in the presence of [³²P]orthophosphate showed that phosphoprotein radioactivity increased on stimulation to a greater extent in a membrane-rich fraction than in a mitochondria-rich fraction, these being obtained by immediate density-gradient fractionation of the tissue dispersion. With fractions isolated by differential centrifuging the percentage increase in a combined mitochondrial and nuclear fraction was 5% as compared with 24% (P<0·02) in the microsomal fraction and 30% in the original dispersion before fractionation. The sensitive phosphoprotein therefore appears to be located in structures sedimenting with the microsomal fraction, rather than with the nuclear fraction as previously claimed.     

Interaction of platinum with metallothionein-like ligands in the rat kidney after administration of cis-dichlorodiammine platinum II

After the administration of the anticancer drug cis-dichlorodiammine platinum II (cisplatin) to male rats, the Pt in the soluble fraction of the kidney is isolated, by gel filtration, in association with a high molecular weight component and a low molecular weight fraction. At 24 h, Pt is also recovered in a metallothionein-like fraction which elutes from Sephadex G-50 with a lower apparent molecular weight than endogenous (Cu, Zn)-thionein or Cd-thionein isolated from the kidneys of Cd2+-treated rats. None of these low molecular weight metal-binding fractions binds to Octyl Sepharose CL-4B. On DE-52 ion exchange chromatography, Cd-thionein is resolved into two isometallothioneins whereas the low molecular weight Pt-binding fraction is only partially purified and contains at least six components which elute at higher gradient concentrations than metallothionein. Pretreatment with Cd2+ which stimulates the synthesis of renal and hepatic metallothionein has no effect on the uptake and subcellular distribution of Pt in the liver and kidneys. Cisplatin treatment reduces the concentration of Cu and Zn in the renal metallothionein and other soluble protein fractions in the kidney. When administered to Cd2+-pretreated rats, cisplatin promotes the loss of Zn from the soluble protein fractions but causes the redistribution of Cd from the metallothionein to the high molecular weight fraction and fails to inhibit the Cd2+-induced accumulation of Cu in the kidneys and the binding of Cu to the soluble protein fractions. It is suggested that metallothionein probably does not have a significant role in the renal metabolism of Pt following the administration of cisplatin to rats.     

Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L-thyroxine

Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid hormone-regulated basic proteins inAchlya ambisexualis

We have investigated specific basic proteins in the oomyceteAchlya ambisexualis, strain E87, which are synthesized and/or accumulate in response to the steroid hormone antheridiol. Three hormone-regulated basic proteins were identified. One of the three basic proteins (64 kDa) was seen only in the cytoplasmic fraction, another (36 kDa) appeared unique to the cell wall/cell membrane fraction, while a third basic protein (63 kDa) was found in both cell fractions. Taken together with previous studies, these results indicate that antheridiol regulates a group of at least four distinct proteins in the molecular weight range of 63,000 to 64,000 which differ in their isoelectric points and in their cellular localization.     

Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8,300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1,514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.     

Control by TSH of protein turnover in thyroid subcellular fractions.

The action of TSH on protein turnover in various subcellular fractions has been investigated in dog thyroid slices incubated in vitro. The results suggest a general inhibition by TSH of protein catabolism. Using double labeline (3/ and 14C) of the proteins, an increase of the disappearance of some labeled material from the microsomal fraction in the presence of TSH has been observed. The protein nature of this material has been established by testing its susceptibility to hydrolysis by trypsin. The fact that the microsomal pellet had to be treated by triton X 100 before hydrolysis by trypsin could occur, suggests that the material is probably enclosed in, or protected by membrane vesicles. Its high molecular weight and its ability to be immunoprecipitated by an antithyroglobulin serum suggest that the microsomal protein, the disappearance of which is stimulated by TSH, is thyroglobulin or one of its subunits. It is suggested that our results reflect the acceleration by TSH of the vectorial transfer of thyroglobulin through the membranes of the endoplasmic reticulum to the colloid space.     

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.     

Amino acid incorporation in relation to molecular weight of proteins in young and adult brain

Rates of protein synthesis were studied in immature and adult rat brain tissue. After an amino acid incorporation period, in vivo or in incubated slices from brain, the soluble protein was fractionated according to molecular weight by column chromatography. In examining soluble whole proteins, no direct correlation between molecular weights and synthesis rates could be established; the highest synthesis rates were found in fractions around 70,000 MW and below 10,000. Incorporation into the subunits after fractionation by SDS gel electrophoresis was proportional to subunit molecular weight, with rates of incorporation into the largest subunits being the highest. The results suggest a relationship between turnover rate and structure of subunits of brain proteins.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.