mM Of Putrescine Research Articles

Analysis of carbohydrate-mediated heterogeneity and characterization of N-linked oligosaccharides of glycoproteins by high performance capillary electrophoresis.

Capillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate-mediated microheterogeneity of the recombinant plasminogen activator (rt-PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt-PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N-Oligosaccharides from fetuin, t-PA and alpha 1-acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.

Control of ornithine decarboxylase activity in jute seeds by antizyme

The control of ornithine decarboxylase activity by antizyme was studied during early germination of jute seeds(Corchorus olitorius). When 2 mM of putrescine and spermidine were applied to the germinating medium, the enzyme activity was markedly inhibited (1.7-fold) during 16 h imbibition. This inhibition could be attributed to the formation of an inhibitory protein termed antizyme. The antizyme was partially purified from jute and barley seedlings. The activity of jute ornithine decarboxylase antizyme was weaker than that of barley.

Inhibition of L-arginine iminohydrolase (EC 3.5.3.6) from Tetrahymena thermophila by putrescine and spermidine: feedback control of polyamine biosynthesis.

L-Arginine iminohydrolase (arginine deiminase, ADI) from Tetrahymena thermophila was purified approx. 75-fold by means of gel permeation chromatography. The Km of the purified enzyme for L-arginine was 412 +/- 25 microM and L-ornithine inhibited the reaction competitively with a Ki of 985 +/- 105 microM. D-Ornithine was a weak inhibitor with a Ki of greater than 10mM. The polyamines putrescine and spermidine inhibited ADI incompetitively with a Kii of 2.8mM for putrescine and 4.3mM for spermidine. Since the concentrations required for inhibition were within the range of the normal intracellular polyamine concentrations in Tetrahymena (maximally 14mM putrescine and 4mM spermidine), it is suggested that the polyamine effects on ADI are of regulatory nature. Thus, polyamine biosynthesis in Tetrahymena thermophila is regulated not only on the level of ornithine decarboxylase activity, but also on an earlier step, the supply of ODC with substrates.

Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.

Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo.

Enzymic synthesis of sym-homospermidine in Lathyrus sativus (grass pea) seedlings.

An enzyme catalysing the synthesis of sym-homospermidine from putrescine and NAD+ with concomitant liberation of NH3 was purified 100-fold from Lathyrus sativus (grass pea) seedlings by affinity chromatography on Blue Sepharose. This thiol enzyme had an apparent mol.wt. of 75000 and exhibited Michelis-Menten kinetics with Km 3.0mM for putrescine. The same enzyme activity could also be demonstrated in the crude extracts of sandal (Santalum album) leaves, but with a specific activity 15-fold greater than that in L. sativus seedlings.

Activation of mammalian DNA ligase by polyamines

The activity of calf thymus DNA ligase was stimulated 2- to 3-fold by the addition of putrescine, spermidine and spermine. The optimal concentrations were 5 mM for putrescine, 0.5 mM for spermidine and 0.05–0.1 mM for spermine. Spermidine not only lowered the apparent Km value for Mg 2+ but also elevated the V value, whereas the apparent Km value for ATP was not changed. In the presence of Mn 2+ as a divalent cation, the V value was stimulated 3-fold by 0.5 mM spermidine. Monovalent cations, such as KCl and NaCl could replace the polyamines at the optimal concentrations of 50 mM and diminish the stimulation by the polyamines.

Polyamines and polyamine-metabolizing enzyme activities in human semen

The content of polyamines putrescine, spermidine and spermine as well as the activities of some polyamine-metabolizing enzymes have been analyzed from normal and pathological human semen samples. Seminal fluid from semen samples showing no apparent abnormalities in semen analyses contained about 0.2 mM of putrescine, 0.1 mM of spermidine and 3 mM of spermine. Seminal plasma was found to be an exceptionally rich source of diamine oxidase activity (EC 1.4.3.6). In addition, polyamine-synthesizing enzyme activities, S-adenosylmethionine decarboxylase and spermidine synthase, were invariably found in seminal plasma. Spermatozoa contained very low or undetectable activities of 5-adenosylmethionine decarboxylase and spermidine synthase; however, a definitive diamine oxidase activity was found also in the cellular component of the semen. The activity of diamine oxidase, and especially that of spermidine synthase, was relatively high in semen samples having low sperm density (less than 20 × 10 6 spermatozoa per ml). With increasing number of spermatozoa there was a slight decrease in both activities, but as the sperm count exceeded 100 × 10 6 per ml the activity of diamine oxidase and spermidine synthase sharply increased. The concentration of spermine in seminal plasma showed only minor changes in relation to the number of spermatozoa in semen, the changes being roughly opposite as compared to the changes of the enzyme activities. A significant negative correlation was found between the concentration of putrescine and the activity of diamine oxidase in seminal plasma. Several properties of diamine oxidase from human seminal plasma were found to be comparable to those of other mammalian diamine oxidases. Using radioactive putrescine as the substrate the enzyme activity was inhibited by addition of unlabelled histamine, cadaverine, spermidine and spermine. The enzyme was also powerfully inhibited by various carbonyl reagents and methylglyoxal bis-(guanylhydrazone).