scholarly journals The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1971 ◽  
Vol 123 (5) ◽  
pp. 959-966 ◽  
Author(s):  
J. Hradec ◽  
Z. Dušek ◽  
E. Bermek ◽  
H. Matthaei
Keyword(s):  
Cholesteryl Ester ◽  
Rat Liver ◽  
Enzyme Purification ◽  
Residual Activity ◽  
Normal Function ◽  
Enzymic Activity ◽  
Elongation Factors ◽  
Enzyme Preparations ◽  
Active Transfer ◽  
Peptide Elongation

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.

scholarly journals Decreased activity of peptide-elongation factors after treatment with cholesterol esterase

1978 ◽  
Vol 172 (1) ◽  
pp. 9-13 ◽  
Author(s):  
J Hradec ◽  
Z Tuháčková ◽  
Z Dušek
Keyword(s):  
Phospholipase A2 ◽  
Elongation Factor ◽  
Normal Activity ◽  
Normal Function ◽  
Cholesterol Esterase ◽  
Elongation Factors ◽  
Ribosomal Subunits ◽  
Binding Factor ◽  
Peptide Elongation ◽  
Sepharose 4B

1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.

scholarly journals Comparison of endogenous and exogenous RNA primers of poly(U) polymerase in rat hepatic ribosomes

1979 ◽  
Vol 177 (3) ◽  
pp. 895-902 ◽  
Author(s):  
T T Hayashi ◽  
K MacFarlane
Keyword(s):  
Rat Liver ◽  
Enzyme Purification ◽  
Polymerase Activity ◽  
Enzymic Activity ◽  
Preliminary Purification ◽  
Exogenous Rna ◽  
Primer Rna

The present work indicates that RNA primer requirements for poly(U) polymerase in the free ribosomes of the rat liver depend upon the degree of enzyme purification. The poly(U) polymerase activity obtained from a crude free ribosomal preparation was compared with the enzymic activity of a partially purified enzyme. After preliminary purification, the enzyme was fractionated by chromatography on Sephadex G-150 and CM-cellulose. Our results demonstrate the presence of several forms of poly(U) polymerase activities, some requiring exogenous RNA and others possessing their own endogenous primer RNA.

scholarly journals Anomeric specificity of d-glucose phosphorylation by rat liver glucose-6-phosphatase

1989 ◽  
Vol 261 (2) ◽  
pp. 509-513
Author(s):  
R Ramirez ◽  
D Zähner ◽  
G Marynissen ◽  
A Sener ◽  
W J Malaisse
Keyword(s):  
Rat Liver ◽  
Glycogen Synthesis ◽  
Liver Microsomes ◽  
Diabetic Rats ◽  
Enzymic Activity ◽  
Rat Liver Microsomes ◽  
Maximal Velocity ◽  
Carbamoyl Phosphate ◽  
Glucose Phosphorylation ◽  
Anomeric Specificity

The anomeric specificity of D-glucose phosphorylation by hepatic glucose-6-phosphatase was examined in rat liver microsomes incubated in the presence of carbamoyl phosphate. At 10 degrees C, the Km for the equilibrated hexose and phosphate donor was close to 56 mM and 11 mM, respectively. The enzymic activity, which was increased in diabetic rats, was about 40% lower in untreated than in sonicated microsomes. No anomeric difference in affinity was found in sonicated microsomes. In untreated microsomes, however, the Km for beta-D-glucose was slightly lower than that for alpha-D-glucose. The maximal velocity was higher with beta- than alpha-D-glucose in both untreated and sonicated microsomes. These data indicate that the phosphotransferase activity of glucose-6-phosphatase cannot account for the higher rate of glycolysis and glycogen synthesis found in hepatocytes exposed to alpha- rather than beta-D-glucose.

Comparison of Different Methods for the Determination of Phenylalanine Hydroxylase Activity in Rat Liver and Euglena gracilis

1984 ◽  
Vol 39 (7-8) ◽  
pp. 728-733 ◽  
Author(s):  
Rita M. Fink ◽  
Erich F. Elstner
Keyword(s):  
Rat Liver ◽  
Euglena Gracilis ◽  
Phenylalanine Hydroxylase ◽  
Hplc Method ◽  
Enzymic Activity ◽  
Hydroxylase Activity ◽  
Product Separation ◽  
Tyrosine Concentration ◽  
Layer Chromatography

Abstract Three different methods for the determination of phenylalanine hydroxylase activity have been compared: a) Differential photometric assay of the increase in tyrosine concentration in the presence of phenylalanine; b) Product separation by thin layer chromatography and scintillation counting of the [14C]tyrosine formed;c) HPLC separation and spectrofluorometric quantification of derivatized amino acids. A comparison of the activities of phenylalanine hydroxylase in rat liver and Euglena gracilis clearly showed that only rat liver contains this enzymic activity as shown by methods b) and c) although pseudo-activity of Euglena gracilis preparations was found during the spectrophotometric test a). The HPLC method proved to be the fastest, most reliable and convenient method for direct tyrosine determination and thus for measuring phenylalanine hydroxylase activity.

Re-Investigation of the Protein Structure of Coenzyme B12-Dependent Diol Dehydrase

1987 ◽  
Vol 42 (4) ◽  
pp. 353-359 ◽  
Author(s):  
Katsuyuki Tanizawa ◽  
Nobuyoshi Nakajima ◽  
Tetsuo Toraya ◽  
Hidehiko Tanaka ◽  
Kenji Soda
Keyword(s):  
Protein Structure ◽  
Membrane Fraction ◽  
Enzyme Purification ◽  
Soluble Fraction ◽  
Proteolytic Cleavage ◽  
Subunit Structure ◽  
Coenzyme B12 ◽  
Enzyme Preparations ◽  
Protein Components ◽  
Limited Digestion

We have purified diol dehydrase, an adenosylcobalamin-dependent enzyme, from Klebsiella pneumoniae by two different procedures to re-investigate its protein structure; one including its extraction with detergent from the membrane fraction, and the other consisting of only chromato­graphic separations of the soluble fraction. The enzyme preparations obtained by these two methods were different in the subunit structure, but both are identical in molecular weight, and in enzymological and immunochemical properties. In addition, the enzyme preparation obtained from the membrane fraction dissociated reversibly into two dissimilar protein components (F and S) in the absence of substrate, as did the preparation from the soluble fraction. Although the subunit multiplicity of component S might be partly due to proteolytic cleavage during the enzyme purification as revealed by limited digestion with trypsin, component F is not a product of proteolytic cleavage of component S, but a primordial and essential constituent of the enzyme.

RESPIRATORY AND ENZYMIC CHANGES IN RESPONSE TO STEROID HORMONES

1970 ◽  
Vol 47 (2) ◽  
pp. 159-166 ◽  
Author(s):  
T. DALTON ◽  
R. S. SNART
Keyword(s):  
Rat Liver ◽  
Enzymic Activity ◽  
Toad Bladder ◽  
Target Tissue ◽  
Transaminase Activity ◽  
Bladder Tissue ◽  
Cellular Processes ◽  
Receptor Sites ◽  
Common Effect ◽  
Stimulation Of

SUMMARY Changes in respiration and enzymic activity were measured in toad bladder tissue and homogenates in response to aldosterone and in rat liver tissue and homogenates in response to corticosterone. The results showed that the stimulation of target tissue may be associated with the saturation of two types of receptor sites for these hormones. The mechanism of action of both hormones is discussed in terms of a common effect on the control of respiration of the target tissue, and a second effect on the control of cellular processes such as the entry of sodium into the epithelial cells of toad bladder or increased transaminase activity in rat liver.

scholarly journals Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

1988 ◽  
Vol 250 (1) ◽  
pp. 53-58 ◽  
Author(s):  
F Flamigni ◽  
C Guarnieri ◽  
C M Caldarera
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Effective Concentration ◽  
The Other ◽  
Limited Effect ◽  
Liver Cytosol ◽  
Rat Liver Cytosol ◽  
Dependent Mechanism

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

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