Louise J. Daniel

A colorimetric micro method for the determination of cholinesterase

Abstract 1. 1. A colorimetric micro method for the determination of cholinesterase activity using acetylthiocholine as substrate has been developed. 2. 2. The method is rapid, requires simple laboratory equipment, and is sensitive to small amounts of enzyme; the reaction is followed by measuring the appearance of one of the reaction products (thiocholine). 3. 3. The method has been applied to the determination of the cholinesterase activity in rat brain, in whole blood and blood plasma of dogs, and in the brains of 12-, 15-, 18-, and 21-day-old chick embryos.

Reduced lysis at high concentrations of lysozyme

Abstract 1. 1. An increase in lysozyme concentration at low levels was accompanied by an increased rate of lysis, but at levels above 0.02 mg./ml. there was a pronounced and rapid decrease in activity for every doubling of the enzyme concentration. 2. 2. The inhibition obtained was essentially the same at the three levels of substrate studied. 3. 3. The inhibition at high lysozyme concentrations was greatly reduced by increasing the sodium-ion concentration. 4. 4. Data are presented that suggest the decreased lysozyme activity is due to the formation of a stable enzyme-substrate complex. This complex mixture was found to contain a substantial amount of active lysozyme but no free substrate or sodium ion.

The biosynthesis of the methyl groups of choline in Ochromonas malhamensis

Abstract Cell-free enzyme systems of Ochromonas malhamensis were employed to investigate the biosynthesis of the methyl groups of phosphatidylcholine. The microsomal fraction of the cells exhibited the greatest activity for the incorporation of the C 14 -methyl group from adenosylmethionine. Adenosylmethionine-C 14 H 3 provided the methyl groups of monomethylethanolamine, dimethylethanolamine, and choline. Methyl-C 14 -cobalamin and N 5 -C 14 H 3 -tetrahydrofolate were not direct precursors of cholinemethyl groups. In order for the C 14 -methyl group from these carriers to be incorporated, conditions for methionine synthesis and activation were required.

The Folic Acid Requirement of Chicks for Growth, Feathering and Hemoglobin Formation.∗

Summary The folic acid requirement of chicks for growth, feathering, and hemoglobin formation has been determined using a purified diet containing 2.0 μg of preformed folic acid per 100 g of diet. The total folic acid content of the basal diets as determined microbiologically following incubation with chick liver was 5 μg per 100 g of diet for Experiment 1 and 15 μg for Experiments 2 and 3. The following amounts of folic acid, which include the amount in the basal diet, were required for its several functions: for survival to 6 weeks of age, approximately 25 μg per 100 g; for growth and hemoglobin formation at 4 weeks of age, 45 μg; for growth at 6 weeks, 45 μg, but for hemoglobin formation at 6 weeks, 35 μg; and for feathering at 6 weeks of age, not less than 55 μg per 100 g of diet. In these experiments the addition of succinylsulfathiazole at 1 or 2% of the diet did not have any effect on growth, feathering, or hemoglobin formation in chicks to 6 weeks of age.

Methionine biosynthesis in Ochromonas malhamensis

Abstract Methyltetrahydrofolate-homocysteine methyltransferase (methionine synthetase) has been partially purified (60- to 80-fold) from the soluble fraction of Ochromonas malhamensis cells. Preliminary studies with the enzyme from cells grown on medium containing cyano-B12 sufficient for optimum growth were done with and without air. Under anaerobic conditions coenzyme B12 inhibited methionine formation. In air the coenzyme gave marked stimulation and was much more active than the system under nitrogen. Without coenzyme B12 the activity under nitrogen and in air was similar. With protein prepared from cells grown with limiting cyano-B12, more methionine was synthesized anaerobically than in air when coenzyme B12 was not present. Anaerobically coenzyme B12 had no effect. In air the coenzyme caused very high activity, equivalent to the B12-adequate preparation plus coenzyme B12. Both holoenzyme and apoenzyme are probably present in each preparation, with more of the apoenzyme in the deficient preparation. The adequate enzyme system in air required in addition to coenzyme B12, ATP, Mg2+, adenosylmethionine, and NADH. The major work dealt with the B12-deficient protein. The cofactor requirements were the same as for the adequate system. No other reducing agent was equivalent to NADH. FMNH2 (H2) was less effective; dithiothreitol inhibited. The unique preference for NADH may be related to the synthesis of holoenzyme. In this system coenzyme B12 was the most active B12 compound. Cyano-B12 was somewhat less active, but required the same cofactors. Methyl-B12 was also less active, and at higher levels caused inhibition. This inhibition only occurred in the presence of ATP. Otherwise, the cofactors needed with methyl-B12 were the same, except that a smaller amount of Mg2+ and a higher quantity of adenosylmethionine were needed for maximum activity. Ethyl-B12 was active and propyl-B12 gave some activity at a low level, but inhibited at a higher level. Some of the activity maybe attributed to its protection of the holoenzyme in air, although the magnitude of the activity suggests that there may have been some enzymatic cleavage of the cobalt-propyl bond. The role of ATP is unclear. It was required for maximum utilization of endogenous B12 and added coenzyme B12 and cyano-B12, and it inhibited the use of methyl-B12. It is proposed that ATP may alter protein conformation permitting better spacial relationships when the “natural” B12 coenzyme [formed from coenzyme B12 and cyano-B12 (in vivo or in vitro)]is present, than when methyl-B12 is the source of the B12 compound.

VITAMIN B12 AND THE SYNTHESIS OF THE METHYL GROUPS OF CHOLINE IN OCHROMONAS MALHAMENSIS.

Abstract Vitamin B12-deficient cells of Ochromonas malhamensis were used to investigate the effect of vitamin B12 on choline-methyl group formation. Vitamin B12 stimulated the incorporation of formate-C14 and formaldehyde-C14, but not of methionine-C14H3, into the methyl groups of phospholipid choline. The optimum utilization of formate-C14 occurred when both homocysteine and vitamin B12 were provided. Methyl-C14-cobalamin donated its methyl group for choline synthesis; however, a greater C14 incorporation was realized when homocysteine was provided. The data support the concept that methionine synthesis is an intermediate step in choline-methyl group biosynthesis, and that in Ochromonas as in other organisms, de novo methyl group synthesis is vitamin B12 dependent.

The effect of vitamin B12 on the synthesis of phospholipides and sphingosine in the developing chick embryo.

Abstract Total phospholipide and the three fractions, lecithin, cephalin, and sphingomyelin, were decreased in the vitamin B12-deficient chick embryo. Although growth of the deficient embryo as measured by dry weight or total nitrogen was significantly retarded at 18 and 21 days of age, the total phospholipide content of the embryo at 21 days was reduced to an even greater extent. The three phospholipide fractions were not decreased proportionately in a B12 deficiency. Lecithin decrease was correlated with growth retardation, whereas both cephalin and sphingomyelin were decreased to a greater extent than was growth. A decrease in the synthesis of sphingosine was noted in the B12-deficient embryo. However, the decreased synthesis was directly proportional to growth retardation, indicating that sphingosine synthesis was not a limiting factor for sphingomyelin formation.

The effect of vitamin B12 on the blood serum protein and lipoprotein in the developing chick embryo.

Abstract The blood serum proteins and lipoproteins were studied in vitamin B 12 -deficient and normal chick embryos at 12, 15, 18, and 21 days of age. In the deficient embryo the blood serum proteins showed little change until the time of hatching. At this age the serum protein was decreased significantly, and the β-globulin fraction was correspondingly decreased. The total lipide and lipoproteins of the serum underwent rapid changes during embryonic development. For the B 12 -deficient serum the total lipide and β-lipoproteins were increased, whereas the α-lipoproteins were decreased at all stages of development.

Effect of oxythiamine on the growth of chicks.

Summary Oxythiamine, the 4′-OH analogue of thiamine, has been shown to be an effective thiamine antagonist in the nutrition of the chick. By increasing the level of oxythiamine fed, the chick weight was decreased until typical thiamine-deficiency symptoms occurred and death ensued. The addition of large amounts of thiamine to the diet prevented the toxicity due to oxythiamine, and the parenteral and oral administration of thiamine to oxythiamine-toxic chicks tended to overcome the inhibition. The chick, as well as the mouse, has been shown to be very sensitive to the addition of oxythiamine to the diet. It seems probable, therefore, that oxythiamine is an antagonist of thiamine in the nutrition of all species that require this vitamin as an essential nutrient. Evidence has been presented which indicates that the thiamine requirement of growing White Leghorn cockerels is greater in the absence of sufficient of the unidentified chick growth factors.

Effect of freezing and thawing on choline dehydrogenase

Abstract 1. 1. The freezing of rat liver caused a marked decrease in choline dehydrogenase activity of homogenates and particulate fractions. 2. 2. The addition of diphosphopyridine nucleotide (DPN) to homogenates from frozen-thawed livers restored most of the activity lost. An equal stimulation of activity occurred upon the addition of DPN to particulate matter from fresh and frozen tissue. This stimulation was greater than that shown with fresh liver homogenates. 3. 3. Inhibition of choline dehydrogenase activity was noted when adenosine triphosphate (ATP) was added to preparations from fresh liver. This inhibitory effect was reduced in liver that was frozen. A stimulation by ATP occurred with particulate matter from frozen liver. 4. 4. In no case did ATP restore the choline dehydrogenase activity of the frozen tissue to that found when DPN was added. 5. 5. The possible action of ATP is discussed.