Eight-step Procedure Research Articles

Galactokinase of Vicia faba seeds.

Galactokinase (EC 2.7.1.6) from the dormant seeds of Vicia faba was purified approximately 1300-fold with an 18% recovery through an eight-step procedure. The preparation showed the presence of only minor contaminations as judged by disc-gel electrophoresis. The native enzyme displayed a molecular weight of approximately 60 000 (determined by Sephadex G-100 gel-filtration) and the subunit value was 30 000. The isoelectric point of the enzyme was 5.3 and the amino acid analysis showed high percentage of acidic amino acids. The pH optimum of the enzyme was 7.3 at 25 degrees C. The relative activity for phosphorylating various monosaccharides followed the order, D-galactose greater than 2-deoxy-D-galactose greater than D-galactosamine; D-fucose, L-arabinose, L-galactose and D-glucose were not phosphorylated. Whereas ATP acted as an efficient phosphate donor, ADP, GTP and UTP were unable to act in this capacity. The Km and the V values of the substrates were determined. The metal ion requirement for the enzymic activity followed the order, Mg2+ greater than Co2+ greater than Mn2+ greater than Ni2+ greater than Ca2+. The enzymic reaction was inhibited by heavy metal ions and sulphydryl reagents indicating the participation of -SH group(s) in enzymic catalysis. Product inhibition was observed; galactose 1-phosphate and ADP were competitive and non-competitive inhibitors, respectively. Seed germination showed an increase in galactokinase level up to 24 h followed by a rapid decrease. The level of raffinose and stachyose decreased continually. The galactokinase level was found to be sufficiently high to phosphorylate the liberated galactose. No free galactose was observed at any stage of germination.

Fruit packaging and handling distribution systems: An evaluation method

Fruit and vegetable distribution systems vary in efficiency according to the inherent characteristics of the produce and the packaging and the handling procedures adopted. The maintenance of quality depends on the interrelationships of these components. It is proposed that performance prediction should be used to obtain maximum benefits from the distribution system. To evaluate the performance of a system requires that its elemen be identified and linked so that the interrelationships can be quantified. It is also necessary to establish acceptable limits for quality and losses. A method for predicting system performance is outlined and, for apples, an eight-step procedure for predicting bruising is given. The method is based on energy concepts and energy absorbed by packaged apples serves as the link between fruit, package and handling. The basic approach should be applicable to a wide range of products.

Indole alkaloid biosynthesis: An investigation into leucine as a possible precursor of the monoterpene portion of vindoline

Because of the nonspecific incorporation of acetate into the monoterpenoid portion of the indole alkaloids, the amino acid leucine has been investigated as an alternative precursor. Incorporations of leucine into the alkaloids vindoline and catharanthine in Vinca rosea are found to be 0.07 and 0.02%, respectively, levels comparable with those of acetate. An equation is developed to calculate the quantity of [ 13C]leucine needed to be fed in order to obtain alkaloids with sufficient 13C enrichment to be analysed by 13C nmr spectroscopy. This equation assumes that percentage incorporations do not decrease with increasing quantities of leucine fed, an assumption found to be true. [2- 13C]Leucine was synthesized from [2- 13C]acetic acid in an eight-step procedure in 11.5% overall yield, and fed to Vinca rosea plants. Incorporations into vindoline obtained using both 14C and 13C were in reasonable agreement, and the activity was found to be spread over seven carbon atoms, none of which corresponded to the two atoms at which activity was expected. It is concluded that the leucine → mevalonate → monoterpene route does not operate in indole alkaloid biosynthesis, leaving open the question of the origin of the monoterpene portion of the alkaloids. An attempt to confirm these results by degradation of 14C-labelled alkaloids was unsuccessful, vindoline proving to be unexpectedly resistant to oxidation.

Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.

N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver.

Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes.

An eight-step procedure has been devised for the preparation of homogeneous rabbit reticulocyte IF-M1. Molecular weight determinations based on IF-M1 activity (gel filtration and sucrose density gradient sedimentation) and based on IF-M1 protein (low speed equilibrium sedimentation and sodium dodecyl sulfate gel electrophoresis) indicate that IF-M1 is active as a single polypeptide chain of 65,000 molecular weight. The amino acid composition of IF-M1 has been determined. There appears to be no unique features in the amino acid composition of IF-M1, except perhaps an elevated proline content (6.9 mol %). The catalytic properties of purified IF-M1 were similar to those previously reported by this laboratory for crude preparations of IF-M1. The sensitivity of IF-M1 activity to N-ethylmaleimide and heat (45 degrees) inactivation was tested in two model reactions requiring minimal complementary factors: (a) AUG-directed fMet-tRNAf binding to ribosomes; and (b) poly(U)-directed polyphenylalanine synthesis at 4 mM Mg2+ (IF-M2A, IF-M2B, EF-1, and EF-2 also required). IF-M1 activity proved to be sensitive to both N-ethylmaleimide and temperature (45 degrees). In addition, a contaminant of partially purified IF-M1 preparations has been found which is capable of fMet-tRNAf binding but is inactive in poly(U)-directed polyphenylalanine synthesis at low Mg2+ concentration.