Michael N. Kazarinoff

N-(5′-phospho-4′-pyridoxyl)amines as substrates for pyridoxine (pyridoxamine) 5′-phosphate oxidase☆

Abstract A preparation of pyridoxine (pyridoxamine) 5′-phosphate oxidase, with a specific activity of 9,400 nmoles/hr/mg protein, 10-fold higher than that previously reported, was used to study the oxidation of various N-(5′-phospho-4′-pyridoxyl)amines. Values for K m , from 3.1 × 10 −5 M to 1.6 × 10 −3 M, and for V max , relative to pyridoxamine-P, of 20 to 140% were obtained. Compounds lacking a 5′-phosphate were not substrates, and the enzymic reaction was dependent on the presence of both FMN and O 2 . N-(phosphopyridoxyl)-L-amino acids had lower K ms than the corresponding -D-amino acid compounds. When 1- 14 C-N-(phosphopyridoxyl)glycine was used as a substrate, no 14 CO 2 was evolved, and 1- 14 C-glycine was detected in the incubation mixture.

Induction of ornithine decarboxylase in colon and liver by starvation and refeeding: A comparison of effects on total and holoenzyme

Abstract In rats, short-term starvation and subsequent refecding induced both holo- and total ornithine decarboxylase (EC 4.1.1.17) levels in colon 5-fold over ad lib fed controls. Starvation alone led to a significant decrease in total ornithine decarboxylase level in colon, while no change was seen in the holoenzyme level. There are only two reported short-term chemical inducers of colon ornithine decarboxylase, and this is the first demonstration of dietary induction in this tissue. Liver total ornithine decarboxylase was also induced by starvation-refeeding, but no holoenzyme effects were seen in any treatment.

Effects of acute and chronic protein deprivation on ornithine decarboxylase levels in rat liver and colon

The effects of protein intake on the levels of total and holo-ornithine decarboxylase (ODC) in colon and liver have been examined in male Sprague-Dawley rats fed varying amounts of casein in a defined purified diet. Pair-feeding was used to eliminate effects of food intake per se. In the colon, both total and holo-ODC activity increased in animals fed a low-protein diet (1% casein). In the liver, on the other hand, both total and holo-ODC activity decreased with protein deprivation. The observed changes in both colon and liver appeared to be maximal within several days after institution of the dietary regimen. Refeeding with a high-protein diet (20% casein) led to a dramatic rise in ODC activity in the liver in animals previously fed a low-protein diet. These results may be important in sorting out the effects of diet on the tumorigenic process.

A radiometric assay of pyridoxamine (pyridoxine) 5'-phosphate oxidase.

Abstract A radiometric assay for pyridoxamine 5′-phosphate oxidase (pyridoxamine (pyridoxine) 5′-phosphate:O 2 oxidoreductase (deaminating), EC 1.4.3.5) has been developed utilizing N -(5′-phosphopyridoxyl)[ 3 H]tryptamine. This assay is more sensitive than previously used colorimetric and fluorescent assays for this oxidase and furthermore is applicable to erythrocytes. Tritiated substrate is incubated with an enzyme sample in the presence of excess unlabeled truptamine and the radiolabeled tryptamine product is extracted into toluene and quantitated by liquid scintillation counting.

[26] FMN-cellulose and derivatives and FMN-agarose

Publisher Summary The FMN derivative of cellulose phosphate is made by first treating 10 g of dry riboflavin in 50 ml of anhydrous pyridine with 40 ml of POCI added drop wise with stirring. The mixture, containing riboflavin dichlorophosphate, is kept at 50°C for 3 hours and then added to a suspension of 80 g of dry cellulose phosphate in 600 ml of anhydrous pyridine. The suspension is kept at 50°C for 24 hours with occasional stirring, and the FMN-cellulose phosphate is filtered off and washed as before. The FMN derivative of DEAE-cellulose is made in the same manner as for FMN-cellulose phosphate, but more extensive washing is necessary to remove the last traces of noncovalently bound flavin.

There is an energy cost for catalytic turnover which arises due to enzyme degradation.

Abstract A method for calculating the energy cost per catalytic turnover which results from enzyme synthesis and degradation is presented. Applying the method to published data on 20 rat liver enzymes yields values ranging from 9.48 × 10 −8 to 5.56 ATP per catalytic turnover. The majority of the values are between 10 −5 and 10 −2 ATP per turnover. A possible role for the expenditure of this energy in regulating enzyme activity in particular cases is discussed.

Protocol for Microinjection of Macroplasmodia of Physarum polycephalum

We have used the following protocol over the last 18 months for the microinjection of 35s-labeled proteins into macroplasmodia. Some patience and dexterity is required in filling the needles with material to be injected; considerably more is required during the injection. Uniform dispersion of injected material is verified by visual inspection in the stereo dissecting microscope with Blue Dextran used as a marker dye. Initially, the dye can be seen to be carried by the protoplasmic streaming. 35S-Labeled material is also uniformly dispersed, as evidenced by constant specific activity in randomly chosen pieces of the Plasmodium. Rupture of the Plasmodium is rarely a problem when the injected material is equilibriated in the buffer described, but can occur if the microburet is dialed too rapidly. The use of black nitrocellulose for growing Plasmodia is recommended to enhance the visual contrast. Standing liquid on the membrane or Plasmodium causes problems and should be avoided.