Characteristics of a nuclear protein kinase from rat epididymis

Purified rat epididymal nuclei possess a cyclic AMP-independent protein kinase activity that phosphorylates of casein. The enzymic activity was solubilized by treating intact nuclei with 1 M (NH4)₂SO₄. One major peak of kinase activity was obtained when the solubilized enzyme preparation was subjected to diethylaminoethyl-Sephadex chromatography. The activity of the kinase was dependent on a bivalent metal ion such as Mg²⁺, Co²⁺, Ca²⁺ or Mn²⁺. NaCl (0.3 M) caused a further activation (approx. 200%) of the metal (Co²⁺)-dependent enzyme. The apparentK _m values of the enzyme for casein, ATP and Co²⁺ are approx. 0.6 mg/ml, 10 ΜM and 2.2 mM respectively. The enzyme was maximally active at pH 5.5. The enzyme showed high specificity for phosphorylation of the acidic protein casein but did not phosphorylate basic proteins, such as histones and protamine. The properties of the nuclear protein kinase were clearly different from those of the cytosolic enzymes previously characterized.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Properties of pyruvate kinase from soybean nodule cytosol

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N₂ fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K⁺ and NH₄⁺ concentrations. In the presence of physiological concentrations of K⁺ and high phosphoenolpyruvate (PEP) concentrations, NH₄⁺ inhibited enzyme activity. Comparisons of kinetic parameters (V_max and apparent K_a) for NH₄⁺ and K⁺ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K_a (monovalent cation) and K_m (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH₄⁺.     

In vitro Transcription of Kinetoplast and Nuclear DNA in Kinetoplastida*†

SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH₄)₂SO₄ and 0.13M (NH₄)₂SO₄ respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn²⁺. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.     

Inhibition by α-Amanitin of Adenovirus 12 Replication in Human Embryo Kidney Cells and of Adenovirus Transformation of Hamster Cells

ADENOVIRUS infection of human embryonic kidney (HEK) cultures seems to induce cellular RNA synthesis, which is preceded by a transient increase in the activities of the Mg²⁺-activated and Mn²⁺-(NH₄)₂SO₄-activated DNA dependent RNA polymerases and in the rate of histone acetylation¹. The two polymerase reactions, assayed in isolated cell nuclei, apparently reflect the activities of distinct nucleolar and nucleo-plasmic RNA polymerases^2,3. We were therefore prompted to test the effect of a specific inhibitor of the mammalian DNA-dependent RNA polymerase function, α-amanitin, on the multiplication of adenovirus. α-Amanitin is a bicyclic octapeptide isolated from the poisonous mushroom Amanita phalloides⁴ and which blocks RNA synthesis in intact animals^5,6. Nuclei isolated from the livers of such animals show a reduced activity of the RNA polymerases associated with nucleoplasm^5,6 and the nucleolus⁶.     

Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH₄)₂SO₄ was stable when frozen as small beads in liquid nitrogen and stored at −80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO₂ and Mg²⁺ activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.     

Poly-ADP-ribosylated histones: potent DNA suppressors

When rat liver nuclear chromatin was sonicated in buffer containing 0.35 M (NH₄)SO₄ to release the engaged RNA polymerases, a potent inhibitor was also released. This inhibitor elicited dramatic inhibition of RNA synthesis regardless of whether the free or engaged RNA polymerase was used. On further analysis, it became apparent that the site of inhibition was on the DNA template, not on the enzyme. This inhibitor could be extracted into 0.25 N HCl by the standard procedure for the isolation of histones. This acid-soluble inhibitor, showing typical histone band on gel, was RNase A and DNase I resistant, but was sensitive to both pronase and snake venom phosphodiesterase digestion, as well as to 0.1 N KOH hydrolysis. Furthermore, when [¹⁴C]adenine labeled poly-ADP-ribosylated histones were digested by snake venom phosphodiesterase, the release of radioactivity was in parallel to the loss of inhibitor activity. We conclude that the inhibitor substances are poly-ADP-ribosylated histones and propose that the poly-ADP-ribosylated histones rather than the histones are the natural suppressors of the gene.     

Binding of a Phosphorylated Inhibitor to Ribulose Bisphosphate Carboxylase/Oxygenase during the Night

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured at various times during the purification of the enzyme from leaves of Nicotiana tabacum which were collected either 1 hour before the start of the photoperiod (predawn) or in the middle of the photoperiod (midday). The activity of the enzyme in extracts of the predawn leaves (0.8 units/mg enzyme) was consistently about 2-fold lower than that measured in extracts of midday leaves (1.7 units/mg enzyme). The activity of the predawn enzyme was increased to that of the midday enzyme following removal of CO₂ and Mg²⁺ (deactivation), (NH₄)₂SO₄ precipitation, or incubation in SO₄²⁻ (18 millimolar required for one-half maximal increase). Following purification to >95% homogeneity, the predawn enzyme was found to have ~0.5 moles of bound organic phosphate per mole of enzyme active sites, while the midday enzyme had only ~0.08 moles of bound organic phosphate per mole of enzyme active sites. Deactivation of the predawn enzyme or treatment with 0.2 molar SO₄²⁻ resulted in the removal of most of the bound organic phosphate. These findings support the hypothesis that following the night period about 50% of the enzyme is catalytically inactive because of the tight-binding of a small molecular weight, phosphorylated inhibitor at the active site.     

Control of Flowering in the Grapevine (Vitis vinifera L.): Formation of Inflorescences in Vitro by Isolated Tendrils

Photosynthesis, primary productivity, N content, and N₂ fixation were determined as a function of applied NH₄⁺ in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH₄⁺ was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH₄⁺, and total N content increased between 0 and 2 millimolar NH₄⁺ but did not change significantly with higher NH₄⁺ applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH₄⁺ concentration (16 millimolar). Symbiotic N₂ fixation by inoculated plants was maximal in peas grown with 2 millimolar NH₄⁺; and apparent relative efficiency of N₂ fixation, calculated from C₂H₂ reduction and H₂ evolution, was maximal in the 2 to 4 millimolar NH₄⁺ concentration range. The capacity to fix N₂ through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH₄⁺ concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH₄⁺ concentration uninoculated plants had greater CER, N content, and dry weight.     

Heterogeneity of protein kinase NII from rat liver nuclei

Protein kinase NII from rat liver nuclei was resolved into two fractions, NIIa and NIIb, by DEAE-Sephadex column chromatography. NIIa was eluted at 151 mM (NH₄)₂SO₄ and NIIb at 175 mM. They had an identical molecular size (125,000 daltons, 7.0S) and subunit composition (αα′β₂). However, they showed significantly different Km values and turnover numbers for casein substrate. Furthermore, NIIa was found predominantly as a form bound to the chromatin, while NIIb was in the nucleoplasmic-soluble fraction in addition to the chromatin-bound fraction. These observations suggest that NII consists of a heterogeneous population of at least two molecular species, differing in the activity and functional states in the cell nucleus.     

Isolation and preliminary characterization of a casein kinase from cauliflower nuclei

A casein-type protein kinase has been isolated from cauliflower (Brassica cauliflora Gars.) nuclei and purified to a specific activity of 23,000 units/milligram of protein (1 unit is defined as the transfer of 1 picomole of ³²Pi from γ-[³²P]ATP to substrate per minute at 28 C). The enzyme has a molecular weight of approximately 39,000 as judged by sucrose density gradient sedimentation. The casein kinase requires ATP as the phosphate donor and will phosphorylate casein and phosvitin, but not histones. The enzyme activity is not affected by cAMP or cGMP. The casein kinase appears to be analogous to casein kinases described in other plant and animal systems.     

Isolation of chromatin bearing nascent RNA from nuclei of sea urchin embryos

Sea urchin embryos were labeled with C¹⁴ thymidine and H³ Uridine, the nuclei isolated, and a chromatin preparation partially deproteinized in salt and detergent. After banding this preparation in a Cs₂SO₄ gradient, the nascent RNA is associated with a small fraction of the chromatin at a density lighter than the bulk chromatin.     

Hexose kinases from the plant cytosolic fraction of soybean nodules

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K_m 0.075 millimolar) than fructose (K_m 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K⁺ ions for maximum activity. The enzyme was specific for fructose (apparent K_m 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 ± 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.     

Separation and partial characterization of two ribonucleic Acid polymerases from pea seedlings

Two DNA-dependent RNA polymerases (ribonucleoside triphosphate:RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from pea (Pisum sativum) seedlings. The enzymes were solubilized by sonication in high salt buffer and were separated by chromatography on diethylaminoethyl cellulose using a linear salt gradient. Polymerase I eluted at 0.10 m (NH₄)₂SO₄, accounted for about 10% of the recovered activity and was completely insensitive to α-amanitin. Polymerase II eluted at 0.14 m (NH₄)₂SO₄, accounted for the remaining 90% of recovered activity and was strongly inhibited by α-amanitin. Both enzymes preferred denatured to native DNA as template, both showed an absolute requirement of divalent cation, and both were sensitive to the ionic strength of the assay medium. The developing pea seedling seems a promising system for studies of possible changes in relative activities and roles of multiple RNA polymerases during eukaryotic development.     

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH₄)₂SO₄ precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.     

Properties of ribulose-1,5-bisphosphate carboxylase from dark- and light-exposed soybean leaves

The low activity of ribulose bisphosphate carboxylase from darkened soybean (Glycine max [L.] Merr. cv. Bragg) leaves was not raised to the level of that from leaves in the light by CO₂ and Mg²⁺, even after a 4-h incubation. The extract of darkened leaves, unlike the extract from illuminated leaves, was not fully CO₂/Mg²⁺-activatable after Sephadex gel filtration in the absence of Mg²⁺. (NH₄)₂SO₄ fractionation eliminated the inhibition effect found in the dark extracts resulting in similar rates for the extracts obtained from leaves in the dark and light. Although the V_max values of the gel-filtered extracts from dark and light leaves differed by 3-fold, the K_m(CO₂)-values were the same (12.7 μM), as were the K_m(RuBP)-values (250 μM). These data support the hypothesis that for soybean leaves in the dark a tightly-binding inhibitor renders much of the ribulose bisphosphate carboxylase enzyme catalytically non-functional.     

Extraction and partial characterization of the glycine decarboxylase multienzyme complex from pea leaf mitochondria

Glycine decarboxylase has been successfully solubilized from pea (Pisum sativum) leaf mitochondria as an acetone powder. The enzyme was dependent on added dithiothreitol and pyridoxal phosphate for maximal activity. The enzyme preparation could catalyze the exchange of CO₂ into the carboxyl carbon of glycine, the reverse of the glycine decarboxylase reaction by converting serine, NH₄⁺, and CO₂ into glycine, and ¹⁴CO₂ release from [1-¹⁴C]glycine. The half-maximal concentrations for the glycine-bicarbonate exchange reaction were 1.7 millimolar glycine, 16 millimolar NaH¹⁴CO₂, and 0.006 millimolar pyridoxal phosphate. The enzyme (glycine-bicarbonate exchange reaction) was active in the assay conditions for 1 hour and could be stored for over 1 month. The enzymic mechanism appeared similar to that reported for the enzyme from animals and bacteria but some quantitative differences were noted. These included the tenacity of binding to the mitochondrial membrane, the concentration of pyridoxal phosphate needed for maximum activity, the requirement for dithiothreitol for maximum activity, and the total amount of activity present. Now that this enzyme has been solubilized, a more detailed understanding of this important step in photorespiration should be possible.