Metabolism of histones and non-histone proteins in liver cell nuclei and chromatin from rats of various ages

The metabolism of various classes of histones and nonhistone proteins in intact nuclei and in liver chromatin of albino Wistar rats aged 1, 3, 12 and 24 months, was studied. It was shown that in the course of postnatal development the metabolism of nonhistone proteins extracted with 0.14 M NaCl in murine liver is increased. Later in ontogenesis, the incorporation of labeled precursors into proteins HMG 14 and HMG 17 decreases; the specific radioactivity of proteins HMG 1 + 2 is higher in 3- and 24-month-old animals. The intensity of metabolism of nonhistone proteins and histones is higher within the composition of the chromatin complex than in the intact nucleus at all stages of postnatal development. Among other histone proteins, histones H1 are characterized by the highest level specific radioactivity in rats of all age groups.     

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion.     

A contribution of nonhistone proteins to the conformation of chromatin.

1. Changes in circular dichroism (CD) spectra and thermal melting profiles of guinea pigliver DNA reassociated with histones and/or nonhistone proteins from the cerebral of liver chromatin are described. 2. In the DNA-histone complex, positive ellipiticity in the CD spectrum at 260-300 nm is progressively lod by a red-shift of the crossover point at around 260 nm. DNA in this complex is thermally stabilised to a considerable extent, but not to such a full extent as is shown with DNA in native chromatin. 3. DNA-nonhistone complex in 0.14 M NaCl is, in contrast to DNA-histone complex, not precipitable by centrifugation at 20 000 X g. DNA in this complex shows only a slight reduction in ellipticity at 260-300 nm, and a very weak thermal stabilisation. 4. Characteristics in the CD spectrum of the native chromatin are most satisfactorily reproduced in the DNA-histone-nonhistone complex. These include a large decrease in ellipticity at 260-300 nm, a red-shift of the crossover point at around 260 nm, and a slight negative band at around 305 nm. Also, DNA in this complex is thermally stabilised to the extent comparable with DNA in the native chromatin. 5. Addition of nonhistone proteins to the preformed DNA-histone complex in 3 M urea renders a half of the complex, named DNA-histone(-nonhistone), unprecipitable upon centrifugation at 20 000 X g in 0.14 M NaCl. CD spectrum and thermal melting profile of the precipitable DNA-histone(-nonhistone) complex are similar to those of the DNA-histone-nonhistone complex, while in the unprecipitable DNA-histone(-nonhistone) comples, the ellipticity at 260-300 nm is significantly elevated and the highest melting transition (at 80 degrees C) is lacking. 6. The CD spectrum of native cerebral chromatin closely resembles that of unprecipitable DNA-histone(-nonhistone) complex, while in liver chromatin, the spec.trum is an intermediate between those of the unprecipitable and pn of chromatin by nonhistone proteins. Cerebral nonhistone proteins bind to DNA and to the DNA-histone complex more extensively than liver nonhistone proteins. 7. It is concluded that, although the basic conformation of DNA in native chromatin is determined largely by histones, nonhistone proteins also play an individual role. There is also an indication that nonhistone proteins exert an organ-specific modification of chromatin superstructure.     

Characterization of transport systems for the transfer of 3,4-L-dihydroxyphenylalanine into slices of rat cerebral cortex.

1. Slices of rat cerebral cortex incubated aerobically at 37 degrees C in Krebs-Ringer-bicarbonate solution accumulated 3,4-L-dihydroxyphenylalanine (L-DOPA) against its concentration gradient. With 1 mM L-DOPA in the medium, tissue-water/medium concentration ratios of about 6 : 1 are reached, which are modified by the presence of other amino acids in the medium. 2. Kinetic analysis suggested that L-DOPA influx into brain cells occurred by at least two saturable processes, which show apparent Km values in the range of 10(-3) M and 10(-5) M, respectively. 3. Prior incubation of the slices in Na+-free (choline-containing) medium at 37 degrees C depressed their subsequent uptake of L-DOPA in normal Na+-containing medium; this inhibition did not appear when the preincubation was carried out at 0-4 degrees C. Besides this effect of preincubation, most of L-DOPA influx into brain slices was independent of the actual concentration of Na+ in the medium; the two saturable processes described in this article behaved similarly in this respect. 4. Most of L-DOPA uptake by the high-Km process is mediated by an agency that resembles the Na+-independent L system described in Ehrlich cells (Oxender, D. L. and Christensen, H. N. (1963) J. Biol. Chem. 238, 2686-2699), both in its specificity and in its participation in exchange phenomena. A lesser component of uptake by a type A mediation is also suggested as contributing to the high-Km process . 5. The kinetic and specificity properties of the low-Km process of L-DOPA uptake suggest a similarity between its mediation and that of the high-affinity systems for L-tyrosine and L-tryptophan found in brain tissue preparations (Belin, M. F. and Pujol, J. F. (1973) Experientia 29, 411-413; Bauman, A., Bourgoin, S., Benda, P., Glowinski, J. and Hamon, M. (1971 Brain Res. 66, 253-263).     

Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.     

Some biochemical properties of polyphenol oxidase from celery

Polyphenol oxidase (PPO, EC 1.14.18.1) was extracted from celery roots (Apium graveolens L.) with 0.1 M phosphate buffer, pH 7.0. The PPO was partially purified by (NH4)2SO4 and dialysis. Substrate specificity experiments were carried out with catechol, pyrogallol, L-DOPA, p-cresol, resorcinol, and tyrosine. The Km for pyrogallol, catechol, and L-DOPA were 4.5, 8.3, and 6.2mM, respectively, at 25 degrees C. Data for Vmax/Km values, which represent catalytic efficiency, show that pyrogallol has the highest value. The optimum pH and temperature were determined with catechol, pyrogallol, and L-DOPA. Optimum pH was 7.0 for catechol and L-DOPA, and 7.5 for pyrogallol. Optimum temperatures for maximum PPO activity were 25 degrees C for pyrogallol, 40 degrees C for catechol, and 45 degrees C for L-DOPA. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 60 degrees C. The order of inhibitor effectiveness was: L-cysteine > ascorbic acid > glycine > resorcinol > NaCl.     

Protein disulfide crosslinking stabilizes a polyoma large T antigen-host protein complex on the nuclear matrix

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.     

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.     

Instability of rat liver chromatin and other nuclear non-histone proteins in alkaline solution.

Non-histone proteins from rat liver nuclei and chromatin were shown to be hydrolysed in 0.1M or-1M-NaOH solutions both at 4 degrees and 18 degrees C; 24h in 1M-NaOH at 18 degrees C is sufficient to break down approx. 77% of these proteins to low-molecular-weight peptides. Loss of protein material banding in the region of pH5.5-8.0 has been demonstrated by isoelectric focusing in polyacrylamide gels, and fine high-molecular-weight bands are no longer visible on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results indicate that care must be taken when analysing non-histone-protein fractions to avoid exposure to alkaline pH conditions.     

Activation of the rat liver androgen-receptor complex

Activation of androgen receptor in rat liver cytosol was studied in vitro. The state of activation was judged by binding of [3H] R1881-receptor complex to chromatin. High ionic strength (0.4 M KCl as a final concentration) provoked the binding of [3H] R1881-receptor complex to chromatin at 0 degrees C. At low ionic strength, activation was very slow at 0 degrees C, but was very rapid at 25 degrees C and reached the maximum at 15 min of heating.     

An analysis of the binding of the chick oviduct progesterone-receptor to chromatin.

The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and nontarget tissues responds in a similar manner to saly and cofactors and has the same K(D) (approx. 3.10(-9) M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K(D) and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [3H]progesterone by preincubation at 25 degrees C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.     

Intermediates in the chaperonin-assisted refolding of rhodanese are trapped at low temperature and show a small stoichiometry.

In vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; EC 2.8.1.1) is facilitated by the chaperonin proteins cpn60 and cpn10 from Escherichia coli at 37 degrees C, but the refolding is strongly inhibited at 10 degrees C. In contrast, the unassisted refolding of rhodanese is efficient at 10 degrees C, but the refolding efficiency decreases as the temperature is raised. These observations provided two measures of the cpn60-rhodanese complex. Thus, we monitored either 1) the cpn60-dependent inhibition of spontaneous folding at 10 degrees C or 2) the recovery of active rhodanese in the complete chaperonin system at 25 degrees C, after first forming a cpn60-rhodanese complex at 10 degrees C. These procedures minimized the aggregation of interactive folding intermediates that tend to overestimate the apparent number of cpn60 14-mers in determining the stoichiometry of protein-cpn60 14-mer interactions. Both procedures used here gave results that were consistent with there being 1 rhodanese binding site/cpn60 tetradecamer. This stoichiometry is significantly less than might be expected from the fact that cpn60 is composed of 14 identical subunits, and it may indicate that rhodanese interacts with a restricted region that is formed when the cpn60 tetradecamer is assembled. The ability to stabilize chaperonin-protein complexes that can subsequently be reactivated will aid studies of the mode of action of the ubiquitous chaperonin proteins.     

Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology

The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ethidium bromide staining or 3'-end labelling. Two experiments were performed to determine the effect of ovary holding time and temperature on oocyte chromatin configuration. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 3h and (2) at 20-37 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6, 6-8 or 8-12h. The oocytes were stained with Hoechst stain 33258 and the chromatin configuration was evaluated. Two experiments were performed to determine the effect of ovary holding time and temperature on cumulus oophorus morphology. Ovaries were held at (1) 20-30 or 35-37 degrees C for up to 2h and (2) for 0-2, 2-4, 4-6, and 6-10h at 35-37 degrees C. The cumulus oocyte complex (COC) were retrieved and the cumulus morphology was evaluated. There was no difference in proportion of follicles with non-apoptotic granulosa cells in the two groups below body temperature (20 and 30 degrees C), but more follicles had apoptotic granulosa cells when the ovaries were held at 35-37 degrees C (P < 0.001). Holding ovaries at 30 degrees C for more than 3h increased the proportion of follicles with apoptotic granulosa cells (P < 0.01). When follicles with non-apoptotic granulosa cells were incubated at room temperature, there was no granulosa cell apoptosis in any of the follicles within the first 3h, but at 5h apoptosis was present in the granulosa cells of 22% of the follicles, and 78% of the follicles contained apoptotic granulosa cells at 24h (P < 0.001). The temperature at which the ovaries were held did not influence oocyte chromatin, although there was a tendency towards more condensed chromatin configurations in the groups below body temperature. More denuded and expanded COCs were present in the lower temperature group (P < 0.001). Oocyte chromatin configuration changed after 6h of holding (P < 0.001), and numbers of compact COCs decreased after 2h (P < 0.05). The present studies suggest that equine follicles should be held for no more than 3h at 20-30 degrees C if granulosa cell apoptosis is to be avoided. To avoid changes in cumulus oophorus morphology, ovaries should be held at 35-37 degrees C and for less than 2h before processing, and to avoid oocyte chromatin configuration changes, ovaries should be stored for less than 6h. When ovaries are to be used in oocyte maturation studies, and assuming that (1) CC is the chromatin configuration of choice for oocyte maturation, (2) that presence of granulosa cell apoptosis promotes maturation of the oocyte and (3) that expanded cumulus oocytes are preferable, the present data suggests that ovaries should be stored for 4-6h before oocyte retrieval.     

Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study

125I-human growth hormone (125I-hGH) binds specifically to receptors on cultures human lymphocytes (IM-9). When this process is studied by use of quantitative EM radioautography, under conditions of incubation at 15 degrees C for 5 min, the ligand is localized to the plasma membrane of the cell. At 30 degrees and 37 degrees C, however, 125I-hGH is progressively internalized by the cell as a function of time. The internalized ligand is found predominantly in the Golgi region of the cells, with a five-fold preferential localization to membrane-bounded structures with the morphological and cytochemical characteristics of lysosomes. Up to 59% of these lysosome-like structures are positive for the acid phosphatase reaction under the conditions of incubation at 37 degrees C for 120 min. When the cell associated radioactivity after 15- 120 min of incubation at 37 degrees C is extracted in 1 M acetic acid and filtered on a Sephadex G-100 column, 58-73% of the material elutes as intact hGH. When cells are incubated with 125I-hGH at 37 degrees C for 15-120 min, separated from the incubation medium, and washed and diluted 100-fold, the percent 125I-hGH dissociable decreases as a function of increasing time of incubation. When cells are incubated with 125I-hGH for 15 min at 37 degrees C and the radioactivity that dissociates from the cells during 15-90 min is studied, the labeled material appearing in the incubation medium is progressively degraded as a function of time of incubation. When the dissociation process is studied radioautographically, grains are found both in plasma membrane and intracelluar compartments after 30 min of association, but after 30 and 120 min of dissociation a higher proportion of grains are in the intracellular compartment. After 120 min of association, there is less dissociation from either compartment and a preferential increase of grains in the intracellular compartment. These data suggest that receptor-linked internalization of a polypeptide hormone provides a mechanism that couples degradation of the ligand with loss of the cell surface receptor.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

The non-histone proteins of the rat liver nucleus and their distribution amongst chromatin fractions as produced by nuclease digestion.

The search for proteins involved in maintaining higher order chromatin structures has led to a systematic examination of the non-histone proteins (NHP) of rat liver nuclei in the context of nuclease digestion studies. 40-45% of the 3H-tryptophan labelled NHP originally present could be removed by extensive washing in a "physiological" buffer, incubation at 37 degrees C with or without nuclease and a further wash step. Nuclei at this stage had a remarkably constant NHP content (ca. 0.73 micrograms/micrograms DNA), independent of the degree of digestion with micrococcal nuclease or HaeIII. The solubilized chromatin produced by limited digestion with either nuclease contained 0.3-0.5 microgram NHP/microgram DNA, this value falling to ca. 0.16 after more extensive cleavage. Insoluble chromatin fractions were between 2-fold (very limited digestion) and 16-fold (extensive digestion) richer in NHP than the corresponding soluble fractions. Gel electrophoresis revealed about 12 NHP bands in soluble fractions, the most prominent of M.Wt. 41.400, while the insoluble material had at least 50 components. These properties were independent of whether lysis of nuclei occurred in 0.2 or 50 mM ionic strength. The large disparity in NHP content between complementary soluble and insoluble chromatin fractions is considered in terms of chromatin organization in vivo and the possible role of NHP migration.     

Subcellular distribution of glucocorticoid receptors in mouse fibroblasts.

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.     

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.     

The effect of insulin on amino acid metabolism and glycogen content in isolated liver cells of juvenile coho salmon, Oncorhynchus kisutch

Amino acid transport and [14C]leucine incorporation into liver proteins as well as the secretion of proteins into incubation medium were studied in liver cells isolated from coho salmon (Oncorhynchus kisutch) parr. Pink salmon (Oncorhynchus gorbuscha) or mammalian (bovine) insulin caused a significant increase in TCA-precipitable radioactivity from both cells and incubation medium. The effects appeared at insulin concentration of 10(-8) M with a maximal response at 5 X 10(-8) M. The radioactivity of the TCA-soluble fraction was not changed by insulin. Insulin increased the amount of the non-metabolized amino acid [14C]cycloleucine, in the TCA-soluble fraction of hepatocytes. The glycogen content of hepatocytes was increased in the presence of insulin at 10(-9) M but was not changed from the control value in the presence of insulin at 10(-8) M.