摘要:

(1) By incubation in 0.1 MNaOH for 10 min at room temperature, it is possible to "saponify" some of the methyl carboxylate linkages in bulk yeast tRNA. By incubation withS-adenosyl[Me-14C]methionine and either homologous (yeast) or heterologous (wheat-embryo) enzymes, it is then possible to "re-esterify" the "saponified" tRNA and thereby effect selective labelling at 5-carboxymethyluridine [Me-14C]methyl ester residues. (2) There is also selective labelling at 2-thio-5-carboxymethyluridine [Me-14C]methyl ester residues when "saponified" yeast tRNA is incubated withS-adenosyl[Me-14C]methionine and homologous (but not heterologous) enzymes. (3) When selectively labelled yeast tRNA is hydrolyzed by RNase T1, both 5-carboxymethyluridine [Me-14C]methyl ester and its 2-thio-analogue are released as part of large oligonucleotides, each of which contains roughly 10 nucleotide residues. (4) There are at least three, and possibly four [Me-14C]-methyl ester-containing oligonucleotides released by RNase T1digestion of selectively labelled "saponified" yeast tRNA. A comparison of the chromatographic properties of the different [Me-14C]oligonucleotides suggests that the same 5-carboxymethyluridine residues are probably targets for both homologous and heterologous enzymes. (5) The properties of the selectively labelled oligonucleotides are consistent with the view that some of them probably are derived from yeast tRNA3Glu, tRNA2Lys, and tRNA3Arg, ail of which are known to contain 5-carboxymethyl methyl esters as part of their anticodon sequences.

ISSN:0008-4018    

DOI:10.1139/o75-001

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

A flow method is described for determination of the kinetic parameters (VmandKm) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.

ISSN:0008-4018    

DOI:10.1139/o75-002

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

TN-C was purified from bovine cardiac muscle. In the absence of Ca2+, cardiac TN-C has an intrinsic sedimentation coefficient of 1.93 S and a molecular weight of 18 000 daltons. Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyosin. Circular dichroism (CD) studies indicate that cardiac TN-C undergoes a major conformational Change upon binding Ca2+. A similar response is elicited by Sr2+, whereas Mg2+has a much less pronounced effect. The presence of Mg2+does not alter the net effects of either Ca2+or Sr2+. Cardiac TN-C is rich in acidic amino acid residues. UV absorption, near UV CD, and fluorimetric studies show that the protein lacks tryptophan and has a relatively high phenylalanine to tyrosine ratio. The results of this study invite direct comparisons with results reported for the skeletal muscle analogue of cardiac TN-C.

ISSN:0008-4018    

DOI:10.1139/o75-003

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

1-Deamino-4-Glu-oxytocin (1-β-mercaptopropionic acid – 4-glutamic acid – oxytocin) was synthesized by sequential reduction by sodium in liquid ammonia and oxidation by hydrogen peroxide of the octapeptide derivative,S-benzyl-β-mercaptopropionyl-tyrosyl-isoleucyl-γ-O-benzyl-glutamyl-asparaginyl-S-benzyl-cysteinyl-prolyl-leucyl-glycinamide. The oxidation analogue was isolated and purified by partition chromatography in two different solvent systems followed by exclusion chromatography on Sephadex G-25. It was found to possess approximately 13 I.U. of uterotonic activity, 34 I.U. of milk ejection activity, and 83 I.U. of milk ejection-like activity per milligram, measured on an isolated strip of lactating mouse mammary gland. 1-Deamino-4-Glu-oxytocin was coupled to AH-Sepharose 4B by way of the free γ-carboxyl group of its residue of glutamic acid. The water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride caused the coupling with approximately 70% effectiveness. The resultant peptide–agarose complex had low biological potency in the assay of milk ejection-like activity.

ISSN:0008-4018    

DOI:10.1139/o75-004

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

Insulin injected intravenously caused a rapid, marked decrease in hepatic glucose secretion in the rabbit, as determined by an isotope-dilution procedure. This was associated with a decrease in the concentrations of gluconeogenic intermediates from phosphoenolpyruvate to triose phosphates, inclusive, compatible with inhibition of gluconeogenesis at phosphoenolpyruvate carboxykinase. The concentration of glucose 6-phosphate was unaltered but that of hepatic glucose was reduced. The specific activities of the hexose phosphates, relative to that of liver glucose, were the same in control and insulin-treated animals. These observations can be explained by a decrease in the activity of glucose-6-phosphatase. It is concluded that this enzyme is a control point for hepatic glucose production and is inhibited by insulin.In the rat, insulin produced a rapid fall in blood sugar. The hepatic glucose output remained normal despite a fall in hepatic glucose 6-phosphate concentration during the initial period of insulin action. This suggests that glucose-6-phosphatase activity was increased. Subsequently the concentration of glucose 6-phosphate returned to normal with no change in the rate of glucose production. The data suggest that in the rat, insulin produces a transient increase in glucose-6-phosphatase activity.

ISSN:0008-4018    

DOI:10.1139/o75-005

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

An enzyme preparation from immature corn kernels catalyzes cleavage ofN6-(Δ2-isopentenyl) adenine to give the aldehyde, 3-methyl-2-butenal, as the major side-chain derived product. This product, in the form of the semicarbazone, was identical with an authentic product by several criteria: chromatographic behavior, mass and ultraviolet spectra.

ISSN:0008-4018    

DOI:10.1139/o75-006

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

In the absence of a monoamine oxidase inhibitor, the bulk of intravenously injected radioactively labelled β-phenylethylamine was oxidized to phenylacetic acid. In the presence of pargyline, most of the label in tissues remained as unchanged phenylethylamine; small amounts of labelled phenylethanolamine, tyramine and octopamine were also identified. After intravenous injection of [14C]phenylalanine, only very small amounts of [14C]phenylethylamine could be located in urine and faeces. β-Phenylethylamine became concentrated in all tissues, including brain, following intravenous introduction both in the presence and absence of pargyline. Its clearance from these tissues and from brain regions was very fast.

ISSN:0008-4018    

DOI:10.1139/o75-007

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

The time course of incorporation of radioactivity into liver phosphatidylethanolamine and its precursors was studied following intraportal injection of [1,2-14C]ethanolamine into rats that had been fasted for 24 h and into nonfasted rats. Marked diminution in the labelling of liver phosphatidylethanolamine and CDP-ethanolamine was present in the fasted groups while the radioactivity contained in the ethanolamine and phosphoryl-ethanolamine pools of these livers was increased. The livers of the fasted rats contained significantly higher levels of ethanolamine, phosphorylethanolamine and CDP-ethanolamine while choline and phosphorylcholine were decreased. These changes are identical to those which we have described earlier in the livers of nonfasted rats fed a choline deficient diet.In a further experiment, the duration of the fasting period necessary to bring about these changes was studied. Incorporation of radioactivity into liver phosphatidylethanolamine at 2.5 min following intraportal injection of [1,2-14]ethanolamine declined progressively up to 7.5 h of fasting and did not decrease further after that time. There was a concomitant decrease in labelling of CDP-ethanolamine with retention of radioactivity in ethanolamine and phosphorylethanolamine. These results show that the liver metabolism of ethanolamine is altered quite early in the fasting period and suggest that it may be related in some way to the metabolic response to fasting.

ISSN:0008-4018    

DOI:10.1139/o75-008

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

With partially purified enzyme preparations from cell-free extracts ofPseudomonas fluorescens, 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid are substrates for glucose oxidase (EC 1.1.3.4.) and gluconate dehydrogenase (EC 1.1.99.3), withKmvalues 18.2 mMand 11.8 mM, respectively. The same enzymes that oxidize glucose and gluconic acid probably oxidize 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid. The latter fluorinated carbohydrates and the presumed formation of 3-deoxy-3-fluoro-2-keto-D-gluconic acid, which has been isolated as a calcium salt and characterized, are not substrates for gluconokinase (EC 2.7.1.12). Both 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid act as competitive inhibitors of this enzyme preparation for gluconate, withKivalues 47.5 mMand 14.8 mM, respectively.

ISSN:0008-4018    

DOI:10.1139/o75-009

出版商:NRC Research Press    

年代:1975

数据来源: NRC

摘要:

A procedure for the quantitative evaluation ofm-tyramine in mammalian tissues is described. It involves isolation of the amine by ion-exchange chromatography, followed by conversion to the dansyl derivative, chromatographic separation, and quantitation by the mass spectrometric integrated ion current technique using an isotopically labelled internal standard. The concentrations ofm-tyramine in some tissues of male Wistar rats were (mean ± S.D., nanograms per gram): brain 0.32 ± 0.03, heart 0.44 ± 0.13. kidney 12.6 ± 3.4, liver 0.27 ± 0.04, lung 0.33 ± 0.11, spleen 0.25 ± 0.07, and blood 0.15 ± 0.04.

ISSN:0008-4018    

DOI:10.1139/o75-010

出版商:NRC Research Press    

年代:1975

数据来源: NRC