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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.

Swainsonine, an alpha-mannosidase inhibitor found in the Australian plant Swainsona canescens and in spotted locoweed, was tested for its effect on the degradation of endocytosed proteins in rat liver cells. The compound inhibited the release of proteolytically derived breakdown products from endocytosed glycoproteins but not from nonglycoproteins. Fifty per cent inhibition of the degradation of 125I-asialofetuin occurred at a concentration of swainsonine in the medium of 6 X 10(-7) M. In the presence of the inhibitor, there was an increased cellular accumulation of the glycoproteins corresponding quantitatively to the decreased degradation, so that total uptake was the same in the presence or absence of the inhibitor. The excess 125I-labeled material which accumulated within cells under these conditions was found entirely in the lysosomal peak on Percoll density gradient centrifugation and migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a mobility indistinguishable from that of the original material. Thus, the inhibition of breakdown occurs prior to any conversion of the protein to intermediates of detectably smaller size. In contrast to the results with swainsonine, degradation of both glycoproteins and nonglycoproteins was inhibited by chloroquine and by 3-methyladenine. The findings with 3-methyladenine are in conflict with earlier reports in the literature on the effect of this compound on degradation of endocytosed proteins.

Rat liver lysosomes, isolated from metrizamide gradients by the method of Wattiaux et al. (Wattiaux, R., Wattiaux-de Coninck, S., Ronveaux-Dupol, M.F., and Dubois, F. (1978) J. Cell Biol. 78, 349-368) took up from the medium and degraded several marker protein preparations, viz. 125I-asialofetuin, [35S]methionine-labeled hemoglobin, and [3H]leucine-labeled rat liver cytosol proteins. Rates were indistinguishable for all the markers, indicating that uptake was by a nonspecific process analogous to fluid pinocytosis. No effect of added MgATP or K+ was observed. Lysosomal degradation of all the markers was inhibited by 10(-4) M chloroquine. Swainsonine, on the other hand, at 10(-5) M, inhibited the breakdown only of the glycoprotein, 125I-asialofetuin. In the presence of the inhibitors, there was an accumulation of markers in the lysosomes in amount corresponding to the decreased breakdown, indicating that uptake was unaffected. Degradation and inhibition were measured at pH 7.0, 6.0, and 5.0 with both intact lysosomes and with lysosomes disrupted by the addition of 0.2% Triton X-100. Degradation with intact lysosomes was relatively independent of pH. On the other hand, activity with disrupted lysosomes was negligible at pH 7.0 and rose rapidly with decreasing pH. Inhibition by 10(-4) M chloroquine and 10(-5) M swainsonine with intact lysosomes decreased sharply with decreasing pH and did not occur with disrupted lysosomes.

The uptake of macromolecular markers by fluid pinocytosis in the rat yolk sac was inhibited by glucagon, with half-maximal effect at a hormone concentration of approximately 3 X 10(-8) M. Glucagon had no effect on the cellular distribution of the marker subsequent to its uptake. Rates of uptake promptly returned to normal when the yolk sacs were transferred from a glucagon-containing to a glucagon-free medium. Epinephrine also inhibited, but only at much higher concentrations. The effect of the latter was augmented by theophylline. Insulin (10(-6) M) had no effect when added alone or with an inhibitory level of glucagon (10(-7) M). The presumption that the hormone effect was mediated by cyclic AMP was supported by the findings that the cellular levels of cyclic AMP were elevated in the presence of glucagon and that dibutyryl cyclic AMP could replace glucagon as an effective inhibitor. The conclusion that the hormone effect was on uptake rather than on subsequent regurgitation was based on the linearity of accumulation in both the presence and absence of glucagon and the inability of glucagon to stimulate loss of invertase from preloaded cells. Colchicine and vinblastine also inhibited uptake. This finding and those of others which are discussed suggest the possibility that effects of cyclic nucleotides on certain cell functions may involve their regulation of microtubular status.

The uptake by rat yolk sacs of native invertase and invertase which was deglycosylated by treatment with endo-beta-N-acetylglucosaminidase was compared. The initial rate of uptake of the deglycosylated enzyme was severalfold greater and its accumulation leveled off much earlier than that of the native enzyme. Uptake rates of the deglycosylated and native forms of the enzyme were proportional to their concentration in the medium in the range employed and were inhibited about 85% by 10(-6) M glucagon in both cases. After preloading of yolk sacs with native invertase, the tissue level of activity remained relatively constant over a subsequent 6-h time period, while with the deglycosylated form, activity declined substantially. Since this difference appears not to be attributable to differences in thermal stability, it is suggested that the deglycosylated form of the protein is more susceptible to intracellular proteolytic digestion. In vitro studies on the digestion of these two forms of invertase by trypsin are consistent with this suggestion.