Acid-insoluble Protein Research Articles

Membrane characteristics of adult rat liver parenchymal cells in primary monolayer culture.

Parenchymal cells, isolated from normal adult rat liver (3 x 10(7) cells/g liver) by collagenase perfusion and maintained in nondividing monolayer culture, were employed to investigate cell surface properties of hepatocytes. Membrane transport systems for asialoorosomucoid (A-OM) and methotrexate (MTX) were lost rapidly in culture, whereas induction of tyrosine aminotransferase and transport of alpha-aminoisobutyrate actually increased during the first 3 days. Alterations in the membrane transport systems for A-OM and MTX reflected more generalized modifications of cell surface components induced during primary culture. Thus, the binding of concanavalin A(Con A) and wheat germ agglutinin (WGA) to cultured hepatocytes increased approximately 2-fold between 24 and 96 hr, and the incorportion of radioactive mannose and glucosamine into trichloroacetic acid-insoluble proteins increased 13-fold and 4-fold, respectively. Plasma membranes were isolated from cultured hepatocytes and the major structural proteins and glycoproteins were analyzed by SDS-polyacrylamide gel electrophoresis. Membrane instability between 24 and 96 hr of culture was characterized by time-dependent alterations in specific polypeptides and extensive changes in Con A- and WGA-binding glycoproteins. Although addition of a complex hormone supplement to the medium increased the number of viable cells and sustained A-OM and MTX transport systems for 24 hr, it had no influence on the altered membrane protein and glycoprotein profiles observed in its absence.

Effects of fasting and insulin administration on polyribosome formation in rat epididymal fat cells.

Polyribosomes from rat epididymal fat pads may be prepared in a synthetically active state and high yield by quick-freezing the tissue in liquid nitrogen, followed by grinding to a frozen powder. The number of initiated (active) ribosomes is limiting in a cell-free protein synthesis assay derived from the frozen preparation that measures completion of nascent polypeptide chains. When starved rats are killed 30 min after a single injection of insulin (0.8 unit, subcutaneously) there is a shift in the ribosomal distribution pattern (on sucrose density gradients) in the direction of larger polyribosomes. This is accompanied by a 2-fold increase in protein synthetic activity of fat cell ribosomes as measured in vitro. When isolated epididymal fat cells are treated with insulin, with or without added glucose, a 2-fold increase in the rate of incorporation of [3H]leucine into [3H]leucine into trichloroacetic acid-insoluble protein is noted within 40 min. The parallel increases in the number of initiated ribosomes, in their synthetic activity in vitro, and in the apparent protein synthetic activity of the whole cell indicate a rapidly evolving action of insulin at the level of peptide chain initiation.

In vitro studies on the effect of salicylates on the synthesis of proteins by guinea pig gastric mucosal tissue

In the work reported in this paper we have studied the effect of salicylates on protein synthesis by (A) intact guinea pig gastric mucosa, (B) isolated gastric epithelial cells of guinea pig stomach and (C) cell-free homogenates of the isolated cells. In experiments on intact gastric mucosa, (A), secretion of newly-synthesised proteins into the mucosal media was also investigated and the nature of the effected, secreted proteins examined by isopycnic CsCl gradient fractionation. Results indicate that synthesis of proteins, as assessed by incorporation of l-[U- 14C]leucine into trichloroacetic acid-insoluble proteins, is significantly impaired in the presence of salicylates in all three systems investigated. In experiments using isolated epithelial cells and cell-free homogenates of these cells, the effect was found to be dose-dependent and not associated with a corresponding reduction of the uptake of the precursor from the medium into the acid-soluble intracellular pool. The inhibitory effect of salicylates on protein synthesis was found to be reversible in experiments using both the intact gastric mucosa, as also the isolated epithelial cell preparations. In all experiments it was found to last for the duration of exposure of the tissue to the action of the drug. In experiments using intact gastric mucosa secretion of the newly-synthesised radioactive proteins into the mucosal medium was also impaired by O- acetyl salicylic acid. Isopycnic CsCl gradient fractionation of the secreted proteins did not reveal any qualitative differences between the salicylatetreated samples and the control samples, indicating non-specific inhibition of protein synthesis by the drug.

Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [ 3H]glycine into acid-insoluble protein and of [ 3H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.

Age-related decline in protein synthesis in the rat parotid gland

This study was undertaken to determine whether protein synthesis declines with age in parorid salivary glands. We have examined the level of incorporation of [ 3H]-leucine into protein and the metabolism of [U- 14C]-D-glucose in 2-, 12-, 18-, 24- and 30-month-old rats. The rate of incorporation of [ 3H]-leucine into perchloric acid insoluble protein by gland slices decreases progressively with age from 138.43±5.43 nmole/mg DNA/h at 2 months to 109.77±6.88, 97±4.68, 71.62±4.13 and 58.08±5.61 nmole/mg DNA/h at 12, 18, 24 and 30 months, respectively. The amount of radioactivity in the perchloric acid soluble fraction remains relatively stable at all ages. The ability of gland slices to oxidize [U- 14C]-D-glucose to 14CO 2 and to incorporate radioactivity into the total lipid does not change with age, whereas the incorporation of radioactivity into protein decreases significantly from 45.06±3.55 at 2 months to 21.75±1.30 nmoles glucuse/mg DNA/h at 24 months. Histologically, no significant change is detected in the proportion of secretory cells to ductal cells in the different age groups. In radioautographs of the glands incubated with [ 3H]-leucine, the distribution of cells with grains is similar in all age groups. Lipid droplets occur at the periphery of the lobules and a large number of lipofuscin granules are present in secretory acinar cells of 12-month-old and older rats when viewed in the electron microscope. However, the structural integrity in the cellular organelles is maintained in the acinar cells of all age groups. These results indicate that the ability of parotid gland cells to synthesize protein decreases with age. The decrease reflects specific changes in protein synthesis and not an overall decline in cellular metabolism or a decrease in the number of cells which incorporate leucine.

Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos

The template activity of chromatin from winter wheat embryos gradually increased during germination and was regulated with some nonhistone proteins different from the two major ones, molecular weight 39k and 59k polypeptides, previously reported.To clarify chromosomal proteins which are involved in regulation of template activity of chromatin, we studied the quantitative and qualitative changes in chromosomal proteins. Differences in acid-soluble and acid-insoluble proteins between chromatins from wheat germ and embryos germinated for various times were visualized by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.Nonhistone proteins of 39k, 41k, and 50k molecular weights were specifically present in wheat germ and in 24- or 48-hour germinated wheat embryos, thereafter greatly reduced or finally disappeared. In contrast, nonhistone protein of 37k was absent in germ and in embryos germinated for 24 hours and appeared after 48 hours of germination. Thereafter it was present in abundant amounts in 96-hour germinated winter wheat embryos and in 72-hour germinated spring embryos, corresponding to 7 and 10% of total nonhistone proteins, respectively. Histone H1, especially H1d, was slightly reduced after 48-hour germination, as much as basic nonhistone proteins having electrophoretic mobilities between H1d and H2B. Further-more, similarity and diversity of chromosomal proteins between spring and winter wheat embryos are shown in this study. A subspecies of histone H1c of spring wheat had faster electrophoretic mobility than that of winter wheat.

The cell free synthesis of bovine lutropin β subunit

RNA prepared from bovine pituitary glands directs the synthesis of trichloroacetic acid-insoluble proteins in the wheat germ cell-free system. Antisera specific to bovine lutropin (LH) beta subunit precipitated a product with a molecular weight of approximately 17,000, suggesting a precursor. Its specificity was demonstrated by the failure of antisera to other pituitary hormones, including LH alpha and thyrotropin (TSH) beta, to successfully precipitate it and also by its competition with unlabeled LH β for the beta antisera. The addition of microsomal membranes resulted in the partial processing of this 17,000 dalton protein to a product of higher molecular weight (Mr ∼ 19,000), suggesting glycosylation.

Cell-free synthesis of rat liver zinc-thioneins.

The induction of rat liver zinc-thioneins mRNA was studied in a wheat germ cell-free translation system. Liver poly A rich polysomal RNA was isolated from rats which had been injected with zinc sulfate 5 h previously. These RNA preparations stimulated the incorporation of [35S]cystine into trichloroacetic acid insoluble proteins when assayed in the cell-free synthetic system. The translation products were characterized by Sephadex G-75 chromatography in 8 M urea--50 mM beta-mercaptoethanol, by disc gel electrophoresis in 4 M area--Tris-glycine buffer (pH 9.2), and by peptide fingerprinting with pepsin. These results were identical with authentic rat liver zinc-thioneins. The zinc-thioneins mRNA activity in the control rats, however, was minimal. The stimulation in zinc-thioneins synthesis observed in the cell-free synthesis was similar to the increased synthesis of these polypeptides in vivo.

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins.

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.

Poly(adenosine diphosphate ribose) synthetase activity in nuclei of dividing and of non-dividing but differentiating intestinal epithelial cells

Poly(ADP-ribose) synthetase activity is found in nuclei of regenerating epithelial cells in the lower half of the crypts of guinea-pig small intestine. Nuclei from non-dividing but differentiating and maturing cells in the upper crypts and on the villi contain no more than about 10% of the synthetase activity of lower-crypt cell nuclei. The product in the active nuclei is shown to be 80% poly(ADP-ribosylated) protein and 20% mono(ADP-ribosylated) protein; 60% of thetotal labelled product was attached to acid-soluble proteins (including histones), and 40% to acid-insoluble (non-histone) proteins. The average number of ADP-ribosyl units in the oligomeric chains of the poly(ADP-ribosylated) proteins was 15 but the range of sizes of (ADP-ribose) oligomers attached to nuclear proteins was smaller in villus than in crypt cell nuclei.

Calcium-dependent inhibition of protein synthesis in rat parotid gland

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was dependent on the phenylalanine concentration in the medium and proceeded linearly for up to 3h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187 required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10 micrometer extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200 micrometer. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both alpha-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.

Effect of triethyllead on protein synthesis in rat forebrain

Triethyllead (PbEt 3) inhibited both oxygen utilization and l-[ U- 14C]-leucine incorporation into acid-insoluble protein in a cell suspension prepared from 20-day-old rat forebrain. At the same concentrations of PbEt 3, protein synthesis was inhibited more than respiration. The cell-free protein synthesis studied in the cytopalsmic fraction was not affected by the neurotoxin. Thus, PbEt 3 does not directly interfere with the protein synthesizing systems and the observed inhibition is secondary to the suppression of the tissue oxidative metabolism.

Primary structure of internal peptide VII of T-even bacteriophages

As first shown by Hershey (1955,1957), osmotic rupture of the head of bacteriophage T2 releases, together with the DNA, an acid-insoluble protein fraction and an acid-soluble peptide fraction. Subsequent chromatographic analysis of the acid-soludle fraction of bacteriophage T4-infected Escherichia coli resolved two peptides, designated II and VII, corresponding to the peptide fraction of T2 described by Hershey (Eddleman & Champe, 1966). Both peptides were found to be composed largely of acidic residues with only a limited number of other amino acids. The amino acid compositions predicted minimum molecular weights of 3900 (33 residues) and 2700 (23 residues) for peptides II and VII, respectively, consistent with their retardation on Sephadex G25 (Champe & Eddleman, 1967). The formation of these peptides in T4-infected cells parallels the synthesis of late proteins. Pulse-labelling experiments showed, however, that the internal peptides are derived from acid-insoluble precursors, suggesting the probable involvement of proteolytic cleavage in their formation. The role of these peptides, if any, in phage replication is unclear. Eddleman & Champe (1966) suggested that they may be inactive remnants of a core protein whose elimination is required for assembly. Alternatively, Laemmli & Favre (1973) have proposed that the internal peptides may function to facilitate packaging of the DNA in the phage head.

The uptake and distribution of 35SO 2 in bean ( phaseolus vulgaris L., cv. Pasuljica)

The fate and distribution of foliar-applied [ 35S] sulphur dioxide and its products has been examined in bean plants. Bean leaves rapidly absorbed SO 2 from polluted atmosphere and within a short time interval translocated some of the products to the roots. Accumulation of sulphur dioxide products is greater in chloroplast and microsomal fractions than in mitochondrial fractions indicating that processes taking place in chloroplasts and microsomes might be affected first by a SO 2-polluted atmosphere. The incorporation of 35S into the acid-insoluble proteins confirms that atmospheric sulphur dioxide can rapidly be transformed into organic compounds.

Solubilization of the chromatin-bound estrogen receptor from chicken liver and fractionation on hydroxylapatite.

1. High-affinity estrogen-binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCL/5 M urea and fractionation on hydroxylapatite. Two estrogen-binding proteins are eluted from hydroxylapatite columns by 20mM phosphate (binding protein I) and 200mMphosphate (binding protein II), respectively. 2. The binding protein I is part of a non-histone protein fraction containing acid-soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the smae affinity for estradiol (Kd approximately 10(-9) M). 3. From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the smae affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable. 4. The "soluble nuclear estrogen receptor" extracted from crude liver nucleir of estrogenized chickens by 0.5 M KCL behaves on hydroxylapatite very similarly to salt/urea-dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated. 5. Chromatography of various preparations on Bio-Gel A-1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein-DNA complex, whereas the binding protein I represents the solubilized nucleic-acid-free chromosomal estrogen receptor. The "soluble nuclear receptor" and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio-Gel A-1.5m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.

A new method for the isolation of eukaryotic nuclear proteins

A new two-step fractionation procedure has been developed which rapidly but quantitatively and qualitatively separates HeLa cell nuclear proteins from nuclear DNA and RNA. Purified HeLa cell nuclei, chromatin (prepared by two different procedures), or isolated heterogeneous ribonucleoprotein particles were first dissociated with sodium dodecyl sulfate detergent, and nuclear proteins and nuclear nucleic acid were partitioned after the addition of sodium chloride and chloroform:isoamyl alcohol. When isolated nuclei of HeLa cells (pulse labeled with [ 3H]leucine) were subjected to this fractionation procedure, over 95% of the radiolabeled nuclear protein was found in the combined interphase and organic phase of the extraction mixture. In parallel experiments, over 90% of the 3H-uridine labeled nuclear RNA and 95% of the [ 3H]thymidine labeled nuclear DNA were consistently partitioned into the aqueous phase of the centrifuged suspensions. Acid soluble nuclear proteins (mostly histone), acid insoluble nuclear proteins (nonhistone), or total nuclear proteins were prepared utilizing this procedure and were analyzed by sodium dodecyl sulfate polyacryalmide gel electrophoresis.

Glycogen phosphorylase in Dictyostelium discoideum. II. Synthesis and degradation during differentiation.

A purified preparation of glycogen phosphorylase from Dictyostelium discoideum was used to elicit specific antisera in rabbits. The antisera were used to quantitate the amount of precipitable phosphorylase protein from cell extracts prepared at various stages of the developmental cycle. Following isotope incorporation studies in differentiating cells, the specific radioactivity of enzyme isolated by antibody precipitation was compared to that of acid-insoluble protein. Prior to 5 hours of development, glycogen phosphorylase could not be detected enzymatically or immunologically. Between aggregation and culmination, the rate of enzyme synthesis increased about 6-fold, then decreased to an insignificant value in young sorocarps. The rate of enzyme degradation was negligible during the period of maximal enzyme accumulation, then increased to a peak value of 40% after culmination, coincident with a rapid drop in phosphorylase activity. The data indicated that the increase in glycogen phosphorylase activity during development results from an increase in the rate of enzyme synthesis.

Uneven extraction of protein in Chinese hamster chromosomes during G-staining procedures

To elucidate the role of chromosomal protein in G-band production, changes of protein distribution in chromosomes were studied in situ at each step of G-staining procedures. As a highly specific stain for protein, dansyl Cl was used, which conjugated with amino groups in polypeptide to emit bright fluorescence under UV irradiation, so that the pattern of fluorescence of dansyl-stained chromosomes was expected to reflect the distribution of protein. Uniform fluorescence pattern observed in untreated, dansyl-stained chromosomes indicated even distribution of protein in the ordinary air-dried chromosomes. The pattern of fluorescence representing the distribution of chromosomal protein after pretreatments of G-staining showed brighter outlines of chromatids, reduced fluorescence of chromosome body, and a slight difference in intensity along chromosome arms which corresponded to G-bands. This correspondence was confirmed when Giemsa stain was removed from G-banded chromosomes and the chromosomes were stained with dansyl Cl. The resulting dim fluorescence pattern conformed to G-bands previously observed in the same chromosomes. Similar events were observed in HCl-extracted chromosome slides, although the fluorescence was considerably reduced in this case. Our results inferred that chromosomal protein was partially lost during pretreatments of G-staining, that acid-soluble protein assumed less significant role in G-staining mechanism, and that uneven deprivation of acid-insoluble protein may occur during G-staining procedures.

Regulation of Tyrosine Hydroxylase Activity By Prostaglandin E1 in Guinea Pig Adrenal Gland

The action of prostaglandin E1 on tyrosine hydroxylase activity in adrenal slices of guinea pig was studied. The activity of tyrosine hydroxylase was decreased by the incubation of adrenal slices with prostaglandin E1 at concentrations beyond 2 µg per ml for 2 hours. The activity of tyrosine hydroxylase was stimulated by dibutyryl adenosine 3',5'-monophosphate in slices of guinea pig adrenal glands. Incubation of adrenal slices with the combination of PGE1 and DBc-AMP led to a tyrosine hydroxylase activity higher than that with PGE1 alone, but not as great as DBc-AMP alone. It was suggested that PGE1 inhibited the enzyme activity independently of the cyclic AMP level. Other prostaglandins such as PGA1 and PGB1 were deficient to the extent that the tyrosine hydroxylase activity was decreased. PGE1 inhibited the enzyme activity much to the same extent seen with protein synthesis inhibitors such as cyclo- heximide and actinomycin D. However, PGE1 did not influence the incorporation of L-leucine-14C into acid insoluble protein. The studies reported here showed that PGE1 inhibited the synthesis of tyrosine hydroxylase.

The preparation and preliminary characterisation of chromatin from the slime mould Physarum polycephalum.

A method is described for the preparation of nuclei from plasmodia of the slime mould, Physarum polycephalum. The nuclei can be gently lysed by suspending them in 10 mM EDTA. A method is described for the preparation of chromatin in high yield from nuclei lysed in EDTA. Between 60 and 70% of the total nuclear DNA may be recovered in the chromatin fraction which has the following chemical composition, DNA : RNA : protein = 1.0:1.0:4.0. The acid-soluble protein fraction was present in equal weight proportion to the DNA and consisted almost entirely of the five major Physarum histones resolvable by polyacrylamide-gel electrophoresis in urea. The molecular weights of these histones, determined by polyacrylamide-gel electrophoresis in sodium dodecylsulphate, were as follows: P1, 24500; P3, 14000; lysine-rich P4, 14500; arginine-rich P4, 14500; P5, 13000; P6, 10500. The lysine-rich Physarum histone, P1, differed significantly in molecular weight from the corresponding calf thymus histone, f1 (mol. wt 21000). The acid-insoluble protein fraction from the chromatin was resolved into over 30 different fractions by polyacrylamide-gel electrophoresis in dodecylsulphate. The majority of the proteins had molecular weights between 14000 and 60000. No significant differences were observed between the polyacrylamide-gel patterns of chromatin proteins isolated from chromatin prepared from the synchronised nuclei of plamodia in the middle of S and the middle of the G2 phases of the cell cycle.