Purification and physiological role of a peptide stabilizing factor of rat liver phosphofructokinase.

A substance has been purified to apparent homogeneity from rat liver which, as previously reported (Dunaway, G. A., Jr., and Segal, H. L. (1974) Biochem. Biophys. Res. Commun. 56, 689-696), specifically stabilizes the major liver isozyme of phosphofructokinase (PFK-L2) against thermal inactivation and whose level in vivo changes in parallel with and in precedence to that of the enzyme. Molecular weight determinations gave values around 3,500. Evidence for the peptide nature of the factor includes its correspondence with ninhydrin-positive material on gel filtration and paper electrophoresis and its susceptibility to pronase. Electrophoretic behavior indicated at least one free amino group and several carboxyl groups. Amino acid analysis of the peptide yielded only glutamate, glycine, and half-cystine, in equimolar amounts. However, neither GSH nor GSSG have PFK-L2-stabilizing activity. No free sulfhydryl groups were present. Chemical analysis for tryptophan was also negative. The ultraviolet spectrum confirmed the absence of aromatic amino acids. The spectrum exhibited a characteristic peptide peak at 190 nm with no absorbance beyond 240 nm. The factor is unstable to storage in the cold except in the presence of glucose or dithiothreitol. Sucrose, fructose, and GSH were ineffective in this regard. It was slowly denatured by heat or reduced pH even in the presence of glucose. The factor was induced in fasted animals specifically by glucose, of the nutrients tested, and in diabetic animals by insulin. Induction by both glucose and insulin was blocked by cycloheximide and actinomycin. The time course of the glucose induction was the more rapid of the two with a marked overshoot to 3 times normal levels at 12 hours. Increased levels of the factor preceded the increased levels of PFK-L2 brought about by glucose or insulin administration. Native PFK-L2 was inactivated by lysosomal extracts, and this inactivation was strongly inhibited by the peptide factor. These results are in accord with the proposal that the peptide plays a role in regulating PFK-L2 turnover in vivo. The factor also activated the phosphofructokinase-catalyzed reaction by promoting fructose-6-P binding. This effect is analogous to that of AMP on the kinetics of the reaction; however, the factor effect was additive to that of AMP, and the factor did not reverse inhibition by excess ATP as does AMP. We postulate that the stabilizing factor affects an equilibrium between PFK-L2 conformers in favor of one more resistant to lysosomal and thermal inactivation and with greater affinity for fructose-6-P.     

Effect of a cyclic enkephalin analog on the protein content in the hippocampal neurons of rats during the acquisition of a 2-way avoidance conditioned reflex

The protein content has been determined by means of cytointerferometry in neurons of fields CA-1 and CA-3 of the dorsal hippocampus in rats, which were trained in a conditioned reflex of two-way avoidance (CRTA) with the action of subcutaneously injected enkephalin cyclic analogue (ECA) in a dose 10 mkg. It has been found that after ECA injection the protein content in the neuronal nuclei of the hippocampal CA-3 field reduces. The acceleration of the CRTA elaboration occurring during the action of ECA is accompanied by a drastic increase of the protein content in the neuronal nuclei of the CA-3 field. The ECA administration to the rats of the active control groups to which were presented the same number of unpaired conditioned and unconditioned stimuli as during the CRTA elaboration also enhances the protein content in the neurons of the CA-3 field. The rats of all investigated groups in the neurons of the CA-1 field display no such significant shifts. The conclusion has been drawn that ECA produces a regulating influence on protein metabolism in hippocampal neurons depending on their functional state.     

Biphasic stimulation of cellular calcium concentration by 3,5,3'-triiodothyronine in rat thymocytes

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.     

Regulatory characteristics of amino acid transport in newborn rat renal cortex cells.

The uptaken of alpha-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 degrees C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na+ during the preliminary incubation. Cycloheximide (50 muM) and purimycin (1 mM) as well as alpha-aminoisobutyric acid, glycine and proline (5 mM) in the preincubation buffer also abolish the effect, while actinomycin D (0.8 muM) partially the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in effux. There is no alteration in the apparent transport Km but an increase in the V for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

Liver peptide stabilizing factor protects phosphofructokinase against inactivation by fructose-1,6-bisphosphatase.

Rabbit liver fructose-1,6-bisphosphatase (FDPase) can reversibly inactivate both rabbit muscle and rat liver phosphofructokinases (PFK) under appropriate conditions. The peptide factor which stabilizes rat liver PFK-L₂ against thermal inactivation has now been found to protect both PFKs from inactivation by FDPase. Assay at high ATP (ca. 3 mM) is necessary to demonstrate these reversible changes. In addition, the activation of FDPase by liver cytosol, by oleate plus cytosol, or by oleate plus muscle PFK is lowere about 50% in the presence of peptide factor. These observations suggest an active participation of the peptide factor in regulation of liver glycolysis and gluconeogenesis.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.