Canadian journal of biochemistry最新文献

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.

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.

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 alpha 1, alpha 2, and alpha 3, have identical peptide maps. In vitro, however, only polypeptide alpha 1 is synthesized in a reticulocyte lysate programmed with cardiac RNA. These results, together with the observations indicating that tropomyosin alpha 2 corresponds to a phosphorylated polypeptide, suggest that only tropomyosin alpha 1 corresponds to a primary translational product and that forms alpha 2 and alpha 3 are derived from alpha 1 as a consequence of posttranslational modifications.

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 H1(0). 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.

High field magnetic resonance spectroscopy has been utilized to deduce the primary structure of the glycopeptides from a human myeloma gamma 1-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 alpha Man(1-6) and alpha Man(1-3) arms: (i) with beta Gal(1-4) and alpha NeuNAc(2-6), respectively (33%); (ii) with beta Gal(1-4) and beta Gal(1-4), respectively (45%); and (iii) beta Gal(1-4) and beta GlcNAc(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 beta GlcNAc(1-2) of the alpha Man(1-6) arm and that the sialyltransferase has a preference for the beta Gal(1-4) of the alpha Man(1-3) arm.

Dihydrofolate reductase of Walker 256 carcinosarcoma has been purified to homogeneity by affinity chromatography. The enzyme reduced 28 mumol dihydrofolate (FAH2) . min-1 . mg protein-1 at 22 degrees 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.

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.

Mechanism of ammonia oxidation was studied in the reconstituted system of Nitrosomonas membrane fraction plus the Nitrosomonas cytochrome c554. The cytochrome c554 was reduced by hydroxylamine, hydrazine, and ammonia and the reduced cytochrome was oxidized upon the addition of ammonia or carbon monoxide. The oxidation of carbon monoxide in the presence of hydroxylamine or hydrazine was studied as a possible assay method for ammonia hydroxylase where hydroxylamine or hydrazine was supplying the reducing power required for the hydroxylation of carbon monoxide. The stoichiometry of the reaction, Km values for substrates, and effects of pH and inhibitors were investigated. It is concluded that carbon monoxide, a competitive inhibitor for ammonia oxidation, is an alternate substrate for ammonia hydroxylase using the reduced cytochrome c554 as the reducing power.

Intestinal maltase with a neutral pH optimum exists in both a brush border membrane-bound form and a soluble form in suckling rat intestine. Previous experiments in our laboratory have shown that the soluble enzyme contains a component which binds much more tightly to concanavalin A (ConA) than solubilized forms of the membrane enzyme. We studied the origin of this component by subjecting neutral, soluble maltase activity to chromatography on Sepharose 4B at age 13, 18 (preweaning), and 25 (postweaning) days. At 13 days, two maltase peaks were obtained with approximate molecular weights of 400 000 (peak I) and 150 000 (peak II). Peak II was less prominent at 18 days and was absent at 25 days. At 13 days, the majority of peak I consisted of material which was bound between 0.025 and 0.05 M alpha-methyl mannoside on gradient elution chromatography of ConA-Sepharose. Peak II contained material which eluted between 0.075 and 0.3 M alpha-methyl mannoside. At 25 days, all of the soluble maltase eluted between 0.025 and 0.04 M alpha-methyl mannoside. Peak I and peak II maltases had similar pH optima and Km's for maltase. Peak II maltase had a fourfold greater activity toward glycogen than peak I maltase with approximately the same activity for palatinose, turanose, and trehalose. Both maltases were precipitated by an antibody raised against adult membrane-bound maltase. Soluble maltase with neutral pH activity in the suckling rat intestine, therefore, consists of two immunologically related isozymes which differ in their molecular weight, their binding by ConA, and their specificity for glycogen. The small isozyme disappears at or about the time of weaning.