Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

Effects of polyamines on in vitro phosphorylation and acetylation of histones of the cerebral cortex of rats of various ages

$\text{In}\text{vitro}$ phosphorylation and acetylation of histones and their modulation by spermine and spermidine were studied using slices of cerebral cortex of female rats of various ages. Phosphorylation and acetylation of individual histones decrease with increasing age. Spermine and spermidine have stimulatory effects on both the modifications of specific histones in immature rats. These effects decrease with increasing age. Such changes in covalent modifications of histones may alter gene expression and contribute to the aging process.     

Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol.

$\text{In}\text{vitro}$ phosphorylation and acetylation of nonhistone chromosomal (NHC) proteins and their modulation by Ca⁺⁺ and estradiol were studied by incubating slices of cerebral cortex of 2-, 15- and 84-week female rats with ³²Pi and ¹⁴C-Na-acetate. Phosphorylation pattern of NHC proteins is unique for each age. Ca⁺⁺ and estradiol stimulate phosphorylation of different NHC proteins which is also age-specific. Acetylation of NHC proteins decreases precipitously with age. No unique NHC protein is acetylated preferentially at any age, nor does Ca⁺⁺ stimulate acetylation. Estradiol, however, stimulates acetylation of a few NHC proteins. It is suggested that phosphorylation of NHC proteins and its modulation by effectors may be more important for gene expression than their acetylation.     

Decreased phosphorylation of specific proteins in neostriatal membranes from rats after long-term narcotic exposure

The endogenous phosphorylation of membrane-bound proteins was studied in the neostriata of rats treated for three weeks with incrementing doses of morphine. Fractions containing synaptic membranes were incubated with γ-³²P-ATP. Phosphate incorporation into individual proteins was determined by gel-electrophoresis and autoradiography of SDS-solubilized membranes. At short reaction times (10 sec.), phosphorylation of all the endogenous protein substrates was reduced compared to preparations from placebo treated rats, but this decrease was differential. Phosphorylation of the specific protein bands designated F and H (MW 47,000 and 15–20,000) decreased by 60–70% while that of all the other bands decreased by only 15–30%. At longer incubations (2–5 min.) bands F and H remained depressed, while the phosphorylation of all the other bands had reached control values. The bands whose phosphorylation selectively $\text{decreased}$ after long-term narcotic exposure were identified as the proteins whose phosphorylation was reported previously to $\text{increase}$ after training experience. Modifications induced in the phosphorylation of these specific proteins may play a role in the adaptive responses of brain cells to various environmental and pharmacological stimulations.     

Timing of ubiquitin synthesis and conjugation into protein A24 during the HeLa cell cycle

The phosphorylative modification $\text{in vivo}$ of histones after shortterm (0 to 60 min) isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Analysis of the phosphorylation patterns after the purification and separation of histones by SDS/polyacrylamide gel electrophoresis revealed significantly increased phosphorylation of histones H1-1 and H3 and a decrease of the phosphorylation of histones H1-3, H2A, and H2B. There was no apparent effect of isoproterenol on the net phosphorylation of histones H1-2 and H4. The data suggest an effect of isoproterenol on the phosphorylative modification of glioma cell histones via modulation of nuclear phosphorylating and dephosphorylating activities.     

Polyamine induced changes in the ADP-ribosylation of nuclear proteins from rat liver

Purified rat liver nuclei were incubated $\text{in vitro}$ with [³H]NAD. Altered patterns of ADP-ribosylation of nuclear proteins occurred with 1 mM spermidine or spermine with the latter polyamine causing the greater change. Spermine treated nuclei showed a two-fold increase in ADP-ribose incorporation into H1 histones and a decrease in the other histones. Likewise, the incorporation into the more acidic non-histone nuclear proteins was greater with spermine than spermidine. These results suggest that polyamines may exert a regulatory function by altering the pattern of ADP-ribosylation of both histone and non-histone nuclear proteins.     

Modulation by the ratio S-adenosylmethionineS-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. $\text{S-}\text{Adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio $\text{S-}\text{adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ can modulate phosphorylation of a specific protein.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Studies on erythrocruorin. V. Nuclear magnetic resonance quadrupole relaxation study of sodium, calcium and chloride binding.

Metabolically labeled non-histone chromosomal proteins of high specific activity were fractionated on the basis of their sequential extractability from Krebs II chromatin with urea/salt solutions according to Bekhor et al. (1974a). The binding of each of these NHCP² classes to protein-free DNA and histone-DNA complexes (nucleohistone) was measured and compared to the binding to DNA substituted with 5-bromo-2′-deoxyuridine. After reconstitution of the interacting components, the binding of NHCP and histones was measured according to Scatchard formalism by titration of fixed amounts of DNA with increasing inputs of protein ligands under stringent conditions of 0.25 ionic strength, pH 8.0. Histone binding to either native DNA or BrUrd-substituted DNA was found to be essentially the same. In the presence of histones, the binding for all NHCP classes, except for medium 3 NHCP, was enhanced by an order of magnitude over the binding values for NHCP to DNA in the absence of histones. The binding of NHCP to DNA was thus strongly influenced by histones bound to DNA. A general and significant decrease in histone content in the complexes relative to increased NHCP binding was also apparent, with medium 3 NHCP having the greatest activity to weaken histone interaction with DNA and medium 0 the least. Enhancement in NHCP binding to BrUd-substituted DNA in the presence of histones was decreased to about 50% of the binding to control DNA. The distribution and quantity of DNA binding and non-DNA binding NHCP was also estimated by photochemical attachment to 33% BrUrd-substituted DNA in tryptophan-labeled chromatin and by direct binding assays. We have obtained 30% crosslinking for either histones or NHCP to DNA in stringently formed complexes. In histone-NHCP-DNA complexes, histone crosslinking remained unchanged, while that of NHCP increased to 70%. This is further evidence for a modification in the binding of NHCP to DNA in the presence of histones. The percentage of NHCP crosslinked to DNA in native chromatin ranged from 24% for medium 0 NHCP to 50% for medium 1 and 3 NHCP with an average of 35% for total NHCP. These results plus the direct binding assays indicate that NHCP, in addition to high affinity DNA binding, also interacts non-specifically to DNA and to proteins in chromatin. A mechanism is also being proposed to account for the observed BrUrd effects in chromatin.     

Phosphorylation of Plant H2A Histones

Phosphorylation of wheat (Triticum aestivum) and alfalfa (Medicago sativa) H2A histone variants was examined during early seedling growth. The C-terminal regions of wheat H2A variants contain multiple S-P tetrapeptides (serine-proline adjacent to a pair of basic amino acids) which resemble known phosphorylation sites in histones from other species. Phosphorylation of nucleosomal core histones was assessed by autoradiography of proteins labeled in vivo with ³²Pi and resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylation sites were mapped by cleaving in vivo labeled H2A variants with N-bromosuccinimide. Essentially all phosphorylation of nucleosomal core histones in wheat and alfalfa seedlings occurred within the C-terminal peptides obtained from wheat and alfalfa H2A variants. A hypothesis accounting for the presence of large H2A and H2B histone variants in plants and phosphorylation of plant H2A C-terminal regions is proposed. The utility of S-P tetrapeptides for modulation of DNA-protein interactions is discussed.     

Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins.

Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate-polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35,000 and 45,000 and isoelectric points at pH values of 5.4-6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.     

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000–33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone compostion of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.     

Studies on the mechanism of insulin-stimulated protein phosphorylation in adipocytes

Insulin exerts two types of effects on protein phosphorylation in adipocytes. First, insulin stimulates phosphorylation of a 123,000 dalton peptide (ATP citrate lyase); second, insulin inhibits the epinephrine-stimulated phosphorylation of a 69,000 dalton peptide.Propranolol, nicotinic acid and concanavalin A, agents which, like insulin, inhibit epinephrine-stimulated cAMP accumulation, also inhibit epinephrine-stimulated phosphorylation of the 69,000 dalton peptide. These agents do not, however, stimulate the phosphorylation of the 123,000 dalton peptide. Carbamylcholine and a variety of cyclic nucleotides (other than cyclic AMP and dibutyryl cAMP) do not alter protein phosphorylation in intact adipocytes. Finally, under conditions wherein insulin fails to inhibit dibutyryl cAMP-stimulated phosphorylation of the 69,000 dalton peptide, insulin-stimulated phosphorylation persists.Thus, while insulin $\text{inhibition}$ of epinephrine-stimulated phosphorylation may be mediated by insulin-induced alterations in cAMP accumulation or action, insulin-$\text{stimulated}$ phosphorylation is $\text{not}$ due to alterations in cyclic nucleotide accumulation or action.     

Protein turnover in muscle: Inhibition of the calcium activated proteinase by mersalyl without inhibition of the rate of protein degradation

The relative roles of neutral and lysosomal proteinases in degrading intracellular proteins have been examined in rat gastrocnemius muscle. A comparison of the relative activities of the proteinases shows that cathepsin B is 10 times more active in muscle than the calcium activated proteinase. This dramatic difference suggests that, if the calcium activated proteinase is required for protein degradation, it might be rate limiting. $\text{In}\text{,}\text{vivo}$ rates of protein degradation were measured after pulse labeling with [³H]N-ethylmaleimide. The rates were not diminished by intramuscular injection of mersalyl at concentrations that inhibited the calcium activated proteinase by at least 35% throughout the 72 h period of the experiments. On the other hand, the lysosomal proteinase, cathepsin B, increased after mersalyl treatment to 370% by 72 h. Therefore, we conclude that lysosomes are necessary for the degradation of modified proteins in muscle and we question the role of the calcium activated proteinase in this process.     

The phosphorylation of high mobility group proteins 14 and 17 from Ehrlich ascites and L1210 invitro

The ability of the high mobility group (HMG) proteins to be phosphorylated was examined in Ehrlich ascites and L1210 cells incubated $\text{in}\text{vitro}$. HMG proteins were selectively extracted from isolated nuclei with 2% trichloroacetic acid, and electrophoretically separated on acid-urea or SDS polyacrylamide gels. Autoradiography of the gels revealed that among the HMG proteins, only HMG 14 and 17 were labeled. The specific activities of these two proteins were approximately equal to that of histone H1. Phosphorylation of HMG 14 and 17 reached a maximum in 2–3 hr and had turnover rates in pulse-chase experiments similar to that of phosphorylated histone H1.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

Action of Solanum malacoxylon on calcium metabolism in the rat

The administration of an aqueous extract of the leaves from $\text{Solanum malacoxylon}$ to vitamin D-deficient rats fed a normal calcium, normal phosphorus diet markedly increased serum calcium concentration within 48 hours. The $\text{Solanum malacoxylon}$ extract also stimulated intestinal calcium transport in the vitamin D-deficient rat but was without effect on the mobilization of calcium from bone. The extract from 100 mg of dry $\text{Solanum malacoxylon}$ leaves was more effective than 25 units of vitamin D given daily to vitamin D-deficient rats in stimulating intestinal calcium transport but its effect was not additive to that of the vitamin D. The results demonstrate that the action of $\text{Solanum malacoxylon}$ is independent of vitamin D and, although it can substitute for vitamin D in the stimulation of intestinal calcium transport activity, it cannot substitute for vitamin D in the mobilization of calcium from bone.     

Trilostane,an orally active inhibitor of steroid biosynthesis

Trilostane is a competitive inhibitor of 3β-hydroxysteroid dehydrogenase. $\text{In}$$\text{vitro}$, the drug inhibits conversion of pregnenolone to progesterone but does not alter conversion of cholesterol to pregnenolone nor progesterone to corticoid hormones. When given orally to rats, trilostane inhibits corticosterone and aldosterone production and elevates circulating levels of pregnenolone at doses lower than those that produce adrenal hypertrophy or inhibit gonadal steroidogenesis.