4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and mannose-6-phosphate

Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Antiporters; Diphosphates; Ethylmaleimide; Galactosephosphates; Glucose-6-Phosphatase; Glucose-6-Phosphate; Hydrolysis; Isoenzymes; Liver; Male; Mannosephosphates; Microsomes, Liver; Monosaccharide Transport Proteins; Nuclear Envelope; Phlorhizin; Phosphates; Rats; Rats, Sprague-Dawley; Substrate Specificity

In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose-6-phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent-disrupted brain microsomes to distinguish between glucose-6-phosphate and mannose-6-phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently-linking reagents [e.g., 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Antiporters; Brain; Diazonium Compounds; Glucose-6-Phosphatase; Glucose-6-Phosphate; Glucosephosphates; Hydrolysis; Kinetics; Male; Mannosephosphates; Microsomes; Monosaccharide Transport Proteins; Phosphotransferases; Rats; Rats, Inbred Strains; Sulfanilic Acids

Intact microsomes from groups of fed, fasted, glucocorticoid-treated (triamcinolone) and diabetic (alloxan) rats were reacted with 4,4'-diisothiocyanostilbene-2,2'-disulfonic (DIDS), a specific inhibitor of microsomal glucose-6-P translocase. The concentrations that inhibit by 50% were 41 +/- 2, 31 +/- 1, 39 +/- 4, and 18 +/- 1 microM (mean +/- S.E.; n = 3); (order as above). The maximal levels of inhibition of the translocase by DIDS were 66 +/- 2, 79 +/- 2, 63 +/- 1, and 88 +/- 1%, respectively. The differences in the values for the different groups of animals are statistically significant, except for comparisons between fed and triamcinolone-treated animals. Microsomes from the same groups of animals were treated with the tritiated reduced derivative of DIDS, [3H]H2DIDS, which labels a 54,000-dalton polypeptide, previously implicated as a component of the glucose-6-P translocase. The mean values (+/- S.E.) of [3H]H2DIDS bound to the polypeptide under saturating conditions were 100 +/- 6, 120 +/- 9, 62 +/- 7, and 101 +/- 15 pmol/mg of microsomal protein, respectively. The amount bound in microsomes from triamcinolone-treated rats is significantly lower from the values for the other three physiological states, which do not differ significantly from each other. The presence of glucose-6-P, but not mannose-6-P, during the [3H]H2DIDS reaction significantly stimulates the labeling of the 54,000-dalton polypeptide in microsomes from all the classes of animals above, except the diabetic animals. These results indicate that DIDS and [3H]H2DIDS are probes sensitive enough to discern differences in the translocase due to physiological regulation. On the basis of the labeling studies with [3H]H2DIDS, the increase in translocase activity observed in microsomes from fasted, triamcinolone-treated, and diabetic rats cannot be ascribed to increased numbers of translocase molecules, but rather to increased functional activity of the translocase protein.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antiporters; Diabetes Mellitus, Experimental; Fasting; Glucose-6-Phosphate; Glucosephosphates; Kinetics; Male; Mannosephosphates; Microsomes, Liver; Monosaccharide Transport Proteins; Phosphotransferases; Rats; Rats, Inbred Strains; Stilbenes; Triamcinolone