Relations between enzyme activities connected with energy metabolism and parameters of food energy utilization in young and adult rats. Part 2. Enzyme activities related to alpha-glycerophosphate shuttle in various tissues.

The possible significance of food composition connected with the alpha-glycerophosphate (alpha GP) shuttle, a putative metabolic pathway of energy dissipation, was investigated at the level of enzyme activities. Liver, adipose tissue, slow-twitch and fast-twitch muscle of weaned male Wistar rats fed ad libitum for seven and for forty weeks a normal-protein (NP), a low-protein (LP), and a high-fat (HF) diet were examined. No striking dietary influences on cytosalic (NAD-linked glycerophosphate dehydrogenase, glyceraldehyde-phosphate dehydrogenase) and mitochondrial (succinate dehydrogenase, cytochrome c oxidase) enzyme activities could be detected, but mitochondrial alpha-glycerophosphate dehydrogenase (m-GPDH) showed an about twofold increase of its activity in the liver of LP-fed animals after seven weeks. A relationship between the "gross efficiency of food energy utilization" and tissue m-GPDH levels could not be established in general. The proposed inducing effect of a LP diet on the magnitude of the GP shuttle observed in the liver of young and adult rats seems to be interconnected reciprocally with the degree of metabolic energy dissipation only under the conditions of growth. The calculated capacities of the alpha GP shuttle are compatible with the assumption of its function as an energy dissipating pathway which is restricted in its magnitude.

An improved modification of the biuret method for the determination of protein in turbid materials with high lipid and hemoglobin content.

The quantitative determination of protein by means of the biuret method frequently yields erroneous values, especially when applied to turbid, lipid- and hemoglobin-containing materials. These errors can only partially be abolished either by the addition of detergents or by destroying the Cu-protein complex by KCN addition. It was found that most disturbances were almost completely eliminated after prior precipitation of protein by the addition of Triton X-100 to the solubilizing biuret reagent and absorbance measurements being performed at a wavelength of 572 nm before and after the addition of KCN. Values of protein determinations according to the proposed assay and protein concentrations calculated from Kjeldahl nitrogen determinations have been shown to agree fairly satisfactory. The proposed assay represents a relatively simple and versatile approach for the evaluation of protein concentrations in a variety of materials containing lipids, hemoglobin and of some other turbidities as well.

Studies on NAD+ permeability in intact mitochondria from rabbit reticulocytes.

Functionally intact mitochondria from rabbit reticulocytes are characterized by a low NAD+ level after the preparation (0.29 nmoles NAD+ + NADH/mg protein). They are apparently impermeable for NADH and exhibit a slow net uptake of NAD+. From the increase of O2-uptake in state 3 and the increase of NADH concentration in state 4 of respiration after the addition of NAD+ we concluded that 3--10 min are necessary for the saturation with NAD+ at 23 degrees C. 2mM NAD+ extramitochondrially are not sufficient to saturate the mitochondria with NADH and probably NAD+, too. Because of the net uptake of NAD+ we assume that reticulocyte mitochondria lose NAD+ during their preparation. If they are incubated with the physiological concentration of 300 micrometer NAD+, which was found in reticulocytes, a value of 1.9 nmoles NAD+ + NADH mg protein was calculated. At an extramitochondrial NAD+ concentration of 300 micrometer, reticulocyte mitochondria exhibit an almost maximal O2-uptake in the presence of oxaloacetate or alpha-ketoglutarate. It is concluded that the mitochondria in intact reticulocytes contain the "normal" complement of NAD+ + NADH.