Canadian Journal of Biochemistry
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Published By Canadian Science Publishing

0008-4018

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.


1982 ◽  
Vol 60 (12) ◽  
pp. 1116-1122 ◽  
Author(s):  
Magdalena Segura ◽  
Elizabeth Palmer ◽  
José L. Saborío

Cardiac tropomyosin from 20-day-old chick embryos is composed of three different polypeptides with the same molecular weight but different isoelectric points. These polypeptides, which are designated as α1, α2, and α3, have identical peptide maps. In vitro, however, only polypeptide α1 is synthesized in a reticulocyte lysate programmed with cardiac RNA. These results, together with the observations indicating that tropomyosin α2 corresponds to a phosphorylated polypeptide, suggest that only tropomyosin α1 corresponds to a primary translational product and that forms α2 and α3 are derived from α1 as a consequence of posttranslational modifications.


1982 ◽  
Vol 60 (12) ◽  
pp. 1085-1094 ◽  
Author(s):  
R. J. Aubin ◽  
V. T. Dam ◽  
J. Miclette ◽  
Y. Brousseau ◽  
A. Huletsky ◽  
...  

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P] ADP-ribosylated histones on first-dimension acid–urea or acid–urea–Triton gels and on second-dimension acid – urea – cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H10. Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.


1982 ◽  
Vol 60 (12) ◽  
pp. 1095-1100 ◽  
Author(s):  
Yew Phew See ◽  
Bernard R. Glick

An Escherichia coli coupled transcription–translation cell-free system, which is efficient in the synthesis of proteins directed by exogenously added DNA, is described. These cell-free extracts direct protein synthesis against a low background of endogenous protein synthesis providing a means for analyzing the expression of isolated genes. This is especially important when using restriction enzyme-linearized DNAs which are less efficient templates than circular DNAs. This cell-free system has been used to study the expression of the proteins coded by plasmids pBR322 and pBL101.


1982 ◽  
Vol 60 (12) ◽  
pp. 1132-1142 ◽  
Author(s):  
Susan J. Johnson ◽  
Sagar V. Gupta ◽  
Kenneth J. Stevenson ◽  
James H. Freisheim

Dihydrofolate reductase of Walker 256 carcinosarcoma has been purified to homogeneity by affinity chromatography. The enzyme reduced 28 μmol dihydrofolate (FAH2)∙min−1∙mg protein−1 at 22 °C and pH 7.3. Km values with respect to FAH2 and NADPH were 21 and 29 mM, respectively. The IC50 (amount of inhibitor required for 50% loss of enzyme activity) values were 0.2 nM for MTX and aminopterin and 50 and 67 nM, respectively, for N10-formyl FA and triazinate (NSC-139105). The pH maximum is around pH 7.0 and the isoelectric point is 6.8. This reductase has an apparent molecular weight of 21 500. The N-terminal amino acid is valine and the comparison of the N-terminal 20 residues of this reductase shows very high sequence homology with other mammalian reductases. The enzyme contains two cysteine residues and one of these residues is not involved in catalysis. This reductase has four tryptophan residues and modification of one of these residues leads to loss of activity. The intrinsic circular dichroic (CD) spectrum of this reductase is very different from the CD spectra of reductase of Escherichia coli B and L1210/MTX. However, the CD spectra of the enzyme–substrate and enzyme–inhibitor complexes are very similar to that of the L1210/MTX enzyme. This suggests that the ligands may be constrained in similar conformation on the two enzymes. The fluorescence emission maximum at 314 nM when activated at 286 nM is considerably lower than other mammalian enzymes.


1982 ◽  
Vol 60 (12) ◽  
pp. 1109-1115
Author(s):  
Christine Des Rosiers ◽  
Marcel Lalanne ◽  
Joan Willemot

Incubation of freshly isolated rat liver cells with glycerol resulted in an initial decrease, followed by an increase in purine synthesis de novo and in PP-ribose-P availability. The magnitude of these effects was dependent on the concentration of glycerol; as it increased, the initial period of latency or inhibition was prolonged, and the extent of the subsequent stimulation was greater. The intracellular Pi concentration and the [14C]ATP/[14C]ADP ratio were also initially decreased in these cells, and they too returned subsequently to normal values. All these changes were similar to those induced by fructose under the same conditions. The increase in PP-ribose-P availability always preceded that in purine synthesis de novo, indicating that, under most circumstances, PP-ribose-P availability is limiting for purine synthesis de novo. Finally, PP-ribose-P synthesis in these cells varied in parallel with the intracellular Pi concentration and with the ATP/ADP and ATP/AMP ratios.


1982 ◽  
Vol 60 (12) ◽  
pp. 1101-1108 ◽  
Author(s):  
Christine Des Rosiers ◽  
Marcel Lalanne ◽  
Joan Willemot

Prelabeled adenine nucleotides were rapidly degraded in freshly isolated rat liver cells incubated with glycerol at concentrations greater than 0.25 mM. Concomitantly, the [14C]ATP/[14C]ADP and [14C]ATP/[14C]AMP ratios were decreased. There was a transient increase in radioactive IMP, inosine, and hypoxanthine and a constant accumulation of allantoin. Radioactive adenosine also accumulated transiently under certain conditions. The intracellular Pi; concentration was decreased. The magnitude of most of these changes was dependent upon the concentration of glycerol. The effects of glycerol were similar to those of fructose. The extent of endogenous adenine nucleotide catabolism was smaller at high Pi concentration, whereas the extent of glycerol-induced catabolism was unaffected. However, less inosine and more hypoxanthine accumulated during glycerol-induced catabolism at high Pi concentration.


1982 ◽  
Vol 60 (12) ◽  
pp. 1123-1131 ◽  
Author(s):  
A. A. Grey ◽  
S. Narasimhan ◽  
J.-R. Brisson ◽  
H. Schachter ◽  
J. P. Carver

High field magnetic resonance spectroscopy has been utilized to deduce the primary structure of the glycopeptides from a human myeloma γ1-immunoglobulin G (Tem). The major structures found belong to the biantennary complex class of glycopeptides, with a minor (5%) fraction belonging to the bisected biantennary complex class. In the biantennary class, three structures were present with different residues at the termini of the αMan(1-6) and αMan(1-3) arms: (i) with βGal(1-4) and αNeuNAc(2-6), respectively (33%); (ii) with βGal(1-4) and βGal(1-4), respectively (45%); and (iii) βGal(1-4) and βGlcNAc(1-2), respectively (17%). In the bisected biantennary class only the latter termini were found for the two arms. These results suggest that the galactosyl transferase in these cells has a preference for the βGlcNAc(1-2) of the αMan(1-6) arm and that the sialyltransferase has a preference for the βGal(1-4) of the αMan(1-3) arm.


1982 ◽  
Vol 60 (11) ◽  
pp. 1014-1017 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev

A convenient method for the enzymatic preparation of sn-3-[2-3H]phosphatidic acids carrying also 5-, 12-, or 16-nitroxide stearic acids, from sn-3-[2-3H]glycerophosphate and isolated guinea pig liver microsomes, is described in detail. The procedure allows a simultaneous preparation of three spin-labelled sn-3-[2-3H] phosphatidic acids of yields 3–3.5 μmol of each compound which is > 99% pure in respect to the radioactivity and which contains 25 mol% of spin-labelled fatty acids. These phosphatidic acids were approximately equally distributed between the primary and the secondary hydroxyl when 12- or 16-nitroxide stearic acids were used or predominantly (75%) associated with the secondary hydroxyl of sn-3-[2-3H]phosphatidic acid when 5-nitroxide stearic acid was present in the incubation mixture.


1982 ◽  
Vol 60 (11) ◽  
pp. 1025-1031 ◽  
Author(s):  
P. M. Strasberg ◽  
J. A. Lowden ◽  
D. Mahuran

Glucosylceramide:β-glucosidase (glucocerebrosidase, EC 3.2.1.45) has been purified 12 900-fold from human placenta using a specific affinity column. The ligand, glucosyl sphingosine, prepared from glucocerebroside by alkaline hydrolysis, was attached to epoxy-activated Sepharose 6B. The enzyme was applied to the column in citrate–butanol or citrate – ethylene glycol solution at its pH optimum (5.6). No enzyme was bound in the presence of detergent. Glucocerebrosidase was eluted with citrate–taurocholate buffer at low pH or with citrate-taurocholate buffer containing D-gluconolactone at the pH optimum. Citrate–taurocholate solution alone at the pH optimum would not elute the enzyme. The enzyme hydrolyzed both the natural substrate, glucocerebroside, and the artificial substrate, 4-methylumbelliferyl glucopyranoside. Glucocerebrosidase migrated as a single band on 10% sodium dodecyl sulfate–polyacrylamide tube and (or) slab gels, corresponding to a molecular weight of 75 000. It also ran as a single zone of enzyme activity or protein on native gels, composed of 2.2% polyacrylamide – 0.4% agarose containing sodium taurocholate. This is the first reported use of this gel system for the examination of glucocerebrosidase. Overall recovery is 30%. The procedure represents a more rapid and specific technique for purification of glucocerebrosidase than those previously reported.


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