Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.

Antibodies that block the ligand binding site of the cation-dependent mannose 6-phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.

Effect of a 42.2-km footrace and subsequent rest or exercise on muscle glycogen and enzymes.

The purpose of this study was to determine the effect of running a marathon and subsequent rest or exercise during a 7-day recovery period on muscle glycogen, glycogen synthase, hexokinase, and the activity of hexose monophosphate pathway enzymes. Ten well-trained runners were randomly assigned to either a postmarathon rest or exercise group. The rest group (n = 5) performed no physical activity, whereas the exercise group ran at their highest tolerable volitional intensity for 20-45 min during the recovery period. Muscle biopsies were obtained from the gastrocnemius before and after the marathon and 1, 3, 5, and 7 days after the marathon. Muscle glycogen was supercompensated before the marathon (196 mmol glucosyl units kg wet tissue wt-1, mmol X kg-1) and was reduced to 25 mmol X kg-1 after the marathon. Seven days later it was 141 and 109 mmol X kg-1 in the rest and exercise groups, respectively. Measurement of glycogen synthase activity ratio indicated an active enzyme only immediately postmarathon, whereas measurement of glycogen synthase fractional velocity and relative velocity indicated an active enzyme immediately postmarathon and 1 day postmarathon. Hexokinase activity was significantly elevated immediately after the marathon and remained elevated through 5 days postmarathon. The activities of the hexose monophosphate pathway enzymes declined steadily during the experimental period and were significantly reduced 5 and 7 days postmarathon. Low-intensity exercise during the recovery week did not significantly differentiate the exercise group from the rest group.

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium.

We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl beta-galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor IIIGlc of the PTS, lead to a low MglP activity, as measured by methyl beta-galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%-20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MGlP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MGlP synthesis can be relieved in two ways: by externally added cyclic adenosine 3',5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.

Urinary acidification in a patient with glucose-6-phosphate dehydrogenase deficiency. A reevaluation of the role of the hexose monophosphate shunt in renal acid secretion.

The relationship between renal metabolism and urinary acidification is poorly understood. During the past decade evidence has accrued to suggest that the hexose monophosphate (HMP) shunt might serve in the process of urinary acidification by providing reducing equivalents for a redox-coupled membrane-bound proton pump that could transport protons into the tubular lumen. The major support for this hypothesis has come from the finding that HMP shunt activity increases with acute and chronic metabolic acidosis. In the present study, we examine the urinary acidification capacity of a young man with severe erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and with unmeasurable G-6-PD activity in renal cortical tissue. We found that despite unmeasurable G-6-PD activity in renal tissue, the patient was capable of generating a maximally acid urine and increasing total acid secretion. Our findings suggest that the HMP shunt may not be necessary for the urinary acidification process.

Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in beta-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of beta-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme varies directly with its susceptibility to pinocytosis by fibroblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enzyme by treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms, that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.