The question of whether or not a cellular ribonuclease is involved in the cleavage of 16S ribosomal RNA by colicin E3 was investigated. For this purpose ribosomes from strains devoid of some ribonucleases or ribosomes in which ribonucleases had been inactivated by heat or removed by extensive washings were used for the colicin reaction. Since the 16S RNA of all these different ribosomes, and even of the most extensively washed ribosomes, was cleaved by colicin E3, it is suggested that cellular ribonucleases are not involved in colicin E3 action.Thus, colicin E3 seems to be a unique endoribonuclease.Colicin E3 (1) cleaves 16S ribosomal RNA from Escherichia coli into two unequal fragments (2-4). The smaller fragment is about 50 nucleotides long and contains the 3' end of the molecule (2, 3). The cleavage reaction can take place in vivo after infection of sensitive E. coli cells with colicin (2, 3), or in vitro by incubation of ribosomes with colicin (4, 5). The reaction requires the presence of both ribosomal subunits (6, 7). Purified 16S ribosomal RNA is not cleaved by colicin E3 (2, 4). Moreover, colicin E3 has not been reported to possess any other ribonuclease activity.Under the reported conditions for in vitro colicin action on ribosomes (4,(6)(7)(8) there are a number of known, and perhaps several uhknown, ribonucleases that are still attached to the ribosomes. Therefore, it is possible that an adventitious ribonuclease is involved in colicin action. We shall consider here the following possibilities: (a) Colicin itself is the ribonuclease and has a unique substrate specificity; (b) Colicin activates a pre-existing ribonuclease to function as a very specific endoribonuclease; (c) Colicin, in conjunction with a ribosomal protein(s) becomes a ribonuclease, which is an elaboration of the first possibility. The experiments reported here suggest that either possibility a or c is the most likely explanation of colicin action. MATERIALS AND METHODSRibosomes from Escherichia coli strains D10 (8), MRE 600 (9), and N7060 (10) were used for in vitro colicin assays.These strains are all devoid of ribonuclease I (8-10). Strain N7060 is further characterized by a thermolabile ribonuclease II and a modified polynucleotide phosphorylase (10). Highly purified colicin E3 was kindly provided by Dr. D. R. Helinski (this preparation did not have any measurable ribonuclease activity). Colicin E3 activity was assayed according to Herschman and Helinski (11), by use of the colicin-sensitive strain D10. Kodak x-ray film (NS2T, 12.5 X 17.5 cm) was used for autoradiography. Carrier-free H332PO4 (50 mCi/ml) was purchased from New England Nuclear Corp. 156Isolation of Ribosomes from Colicin-Sensitive Cells. Cells of strain D10 were grown at 370 in 500 ml of mimimal medium (per liter: 14.5 g Tris, -100 mg MgSO4, 500 mg NaCl, 150 mg KCl, 500 mg NH4C1, adjusted to pH 7.4 with HCl) containing 0.2% glucose, 25 mg of L-methionine, and 16 mg of KH2PO4. All subsequent steps in the preparation of ribosomes were done at 4°. Cells were gro...
We inve~tlllated th~ influence of the thyroid hormone ~tatus on ill© levels of protein kinase~', C [PKC) and A {PKA) in the soluble fraction of rat liver, The im)nunodelectabl¢ PKC level in hypothyroid liver was elevated ?.7.fold, wherea~ the: phorhol.ester bindin~ capacity and the immunodetectable =.PKC level were increased 2.4. and 2.6.f`old, respectively. Conversely, in hypothyroid ~ivers the abundance of the reliulatory tyl~= 1 and the eat=tlytl¢ subunits of PKA were lowered to 421X, of the euthyi,oid level as d~termined by immunoblottinl; and by measurinB the substrate ~peeifie pho~phorylation rate of PKA. These chan@s in the PKC (rod PKA level= were reversible upon treatment with 0,5 l~tl TJI00 [t body weight for 2~21 days, The thyroid it.tie dependent altera,,ion~ in hepatic PKC and PKA levels may be responsible for the known chanBes in the response of' hcpatocytes to other h r~rmonal stimuli in hypothyroidism, on PKA, no evidence has been presented on the influence of thyroid hormones on the PKC levels in any organ. Protein kinase C is a family of enzymes encompassing at least 7 subspecies, which has been shown to mediate and modulate transmembrane signalling and which is proposed to play a crucial role in governing cell metabolism and growth [9]. Choosing the liver as an important target organ of thyroid hormone action we investigated whether the levels of ~-,/5'-, ~,-PKC and PKA type I or II are altered in the liver cytosol of hypothyroid rats and whether these changes can be reversed by treatment with L-thyroxine (T~). The signal transduction regularly results in the activation of various protein kinases. The phosphorylation of serine, threonine and tyrosine residues by these enzymes has been demonstrated to be a common cellular mechanism for regulating gene transcription, cell division, membrane transport and metabolic pathways [6]. Thyroid hormones have been reported to stimulate the phosphorylation or dephosphorylation of multiple rat liver cytosolic proteins [7]. A 1.5-fold increase in type I cyclic AMP dependent protein kinase (PKA type I) activity in the liver of hyperthyroid compared to hypothyroid rats has been demonstrated by electrophoresis of cytosolic proteins followed by phosphorylation assays [8]. In contrast to the studies MATERIALS AND METHODS2,1, Animals 25 n~ale Wistar rats (100-120 g) were randomized into 5 different treatment groups and were rendered hypothyroid by feeding an iodine depleted diet and by adding 1% KCIO4 to the drinking water for 35 days. 0.107o 6t~-propyl-2-thiouracil was added to the diet for the first 6 days (modified from [2]). Five control rats were maintained on the iodine depleted cllow with 1 ppm potassium iodide added to the drinking water. The free T4 and total L-triiodothyronine serum levels in the hypothyroid group were 1 ± 0.0°70 and 13 ± 4°70 of the euthyroid (iodide substituted) group, respectively. The hypothyroid animals of 4 Of the 5 groups were daily substituted orally with 0.5 ag Ta/IO0 g body weight for 2, 5, 9 and 21 days, respectively. Vehicle...
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