Localization of Particulate Guanylate Cyclase in Plasma Membranes and Microsomes of Rat Liver*

SUMMARY The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5’-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5’-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble typical Michaelis-Menten with respect GTP and had an apparent K, GTP of 35 properties

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers.
Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent K, for GTP of 35 PM. Ca2+ stimulated the soluble activity in the presence of low concentrations of Mn2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg2+ was less effective than Mn*+ as a sole cation, all enzyme fractions were markedly stimulated with Mg2+ in the presence of a low concentration of Mn*f. Triton X-100 increased the activity of particulate * These studies were supported by a grant from the National fractions about 3-to IO-fold and increased the soluble activity 50 to 100%.
Recently our laboratory (l-3) and Chrisman el al. (4) rcported that rat tissues have two forms of guanylate cyclase (EC 4.6.1.2) that catalyze the formation of guanosine 3':5'monophosphate from GTP. The soluble and particulate enzymes from various rat tissues have different kinetic properties and molecular sizes. In some tissues such as lung and liver most of the enzymatic activity is found in soluble fractions of homogenates (l-11), and most of the early studies have been confined to soluble guanylate cyclasc. However, in all tissues esamined to date significant quantities of the enzyme have been found in particulate fractions (1). In tissues such as heart, intestinal mucosa, cerebral cortex, cerebellum, and others, the majority of the activity is particulate (l-3, 6, 11). The precise subcellular localization of the particulate activity has not been determined. In order to obtain a better understanding of the metabolism of cyclic GLLIP' in tissues and its possible regulation, we determined the cellular localization of guanylate cyclase in rat liver. Although rat liver has only 15 to 30% of the guanylate cyclasc activity in particulate fractions of homogenates, we chose this tissue for these studies because of the previous work by a number of laboratories in developing techniques to separate liver cellular components (12-14). We found the particulate enzyme located in both plasma membranes and microsomes that were characterized with various marker enzymes and electron microscopy. Some of the properties of the particulate enzymes are also described and compared to those of the soluble fraction. Some of these observations have been reported previously in abstract form (15). Fluoride-stimulated adenylate cyclase activity was also determined.
Reaction mixtures were the same as those for guanylate cyclase except that 1 mM ATP and 4 mM M&l2 were used instead of GTP and MnC12.
NaF was 10 mM in all assays. After incubation at 37" for 10 to 20 min. reactions were terminated bv adding 0. FIG. 1. Fractionation with a discontinuous sucrose gradient under "Materials and Methods." Activities of guanylate cyclase, of a crude nuclear fraction from a rat liver homogenate. Fractions 5'-nucleotidase, succinic dehydrogenase, and protein are shown. were obtained after discontinuous sucrose gradient centrifugation The three major peaks of activity (A, B, C) were pooled as desigof a crude nuclear fraction of a rat liver homogenate as described nated for further analyses.
heart (l), lung, and small intestinal mucosa* in which most of the particulate guanylate cyclase activity was found in the crude nuclear fraction, particulate activity was found predominantly in crude nuclear and microsomal fractions of liver. 5'-Nucleotidase and adenylate cyclase were also found in crude nuclear and microsomal fractions (Table I), and these findings are consistent with previous reports (21, 26-28). The latter enzymes have been used as markers for plasma membranes although, as discussed below, these enzymes exist in other cell components as well. Glucose-6-phosphatase was found predominantly in the crude microsomal fraction. Succinic dehydrogenase, an enzyme marker for mitochondria, was found in both crude nuclear and mitochondrial fractions. Small amounts of guanylate cyclase, 5'-nucleotidase, adenylate cyclase, and glucose-6phosphatase were also associated with the crude mitochondrial fraction. These observations suggested that the mitochondrial fraction contained some contaminating cell components. We have not further examined this crude fraction.
Purified plasma membranes were prepared from the crude nuclear and microsomal fractions by the method of Touster et al. (14) with a slight modification.
Since significant amounts of 5'-nucleotidase and adenylate cyclase activities were found near the interface of the 57 y0 and 34% sucrose layers, a layer of 40% sucrose was added to the discontinuous gradient when centrifuging crude microsomal fractions (see under "Materials and Methods"). After discontinuous sucrose gradient centrifugation of crude nuclear fractions, we obtained three peaks of guanylate cyclase and 5'-nucleotidase activities (Fractions A, B, and C, Fig. l), two peaks of succinic dehydrogenase activity (Fractions B and C, Fig. l), and two small peaks of glucose-6-phosphatase activity (Fractions B and C, not shown). Guanylate cyclase activity in the crude nuclear fraction was about equally distributed in the three pooled fractions, Fractions A, B, andC (Table II). The greatest purification occurred in Fraction A where the specific activity increased 7.4-fold. The membrane marker enzymes used, 5'-nucleotidase and adenylate cyclase, were also purified 9.3-fold in Fraction A (Table II). Recoveries of activities of guanylate cyclase, 5'-nucleotidase, and adenylate cyclase in Fraction A were also similar. 5'-Nucleotidase in Fraction A was purified 32-fold from the homogenate with a recovery of 15%. The activity of 5'-nucleotidase in Fraction A was also similar to the report of Touster et al. (14). The purification of adenylate cyclase from homogenates of 11.7-fold with 5.4% recovery in Fraction A was comparable to that of Pohl et al. (29). These investigators obtained a I3-fold purification with 8% recovery with the use of a different technique for preparing liver plasma membranes. We cannot compare the degree of purification of guanylate cyclase from the homogenate to that of 5'-nucleotidase or adenylate cyclase since most of guanylate cyclase in liver is soluble. These studies indicated that Fraction A was primarily plasma membranes. Glucose-6-phosphatase and succinic dehydrogenase were found primarily in Fractions B and C; each fraction contained 2 to 6% of the homogenate activity. When the nuclei were prepared from the 750 x g pellet by the method of Maggio et al. (30), less than 10% of the guanylate cyclase activity with less specific activity was recovered in the sedimentable fraction. From these results we concluded that particulate guanylate cyclase in the crude nuclear fraction of liver homogenates is predominantly in plasma membranes. The activity in other fractions could represent activity in other structures or their contamination with plasma membranes.
Three peaks each of guanylate cyclase, 5'-nucleotidase, and protein were obtained (Fractions A, B, and C) when crude microsomal fractions were examined with discontinuous sucrose gradients (Fig. 2). Two peaks of glucose-6-phosphatase activity (Fractions B and C) were obtained. The activities recovered and the degree of purification for guanylate cyclase, adenylate cyclase, and 5'-nucleotidase were very similar to one another in Fractions A and B (Table II). However, more adenylate cyclase activity was recovered in Fractions B and C than would be predicted from previous reports (28,29). From the original homogenate 5'.nucleotidase and adenylate cyclase were purified 2% and ll%fold, respectively, in Fraction A (not shown) which is comparable to previous reports (14, 29). Fraction C contained 58.4% of the guanylate cyclase, 69.2% of the glucose-6-phosphatase, and 17.2y0 of the 5'-nucleotidase from the crude microsomal fraction, FIG. 2. Fractionat,ion with a discont,inuous sucrose gradient of a crude microsomal fraction from a rat liver homogenat,c. Frac-as described under "Materials and Methods." Activities of guanyltionswcre obt'ained after discontinuous sucrose gradient centrif-ate cyclase, 5'-nucleotidase, glucose-G-phosphatasc, and protein ugation of a crude microsomal fraction of a rat liver homogenate are shown. Thr three major peaks of activity (A, I?, C) were pooled as designated for further analyses. and no purification was achieved. The high recoveries of guanylate cyclase and glucose-6-phosphatase compared to that' of 5'nuclcotidase indicated that' guanylate cyclase was also present, in microsomes (see below). Small amounts of succinic dehydrogenase activit'y were present in Fractions B and C, and only one-t'hird of the act,ivitg in the crude microsomal fraction was recovered. From these result,s it was evident that particulate guanylate cyclase of rat' liver was localized in both plasma menbranes and microsomes.
The conclusions on the basis of biochemical studies are supported by t'he electron microscopic examination of fractions obtained after discontinuous sucrose gradient ccrurifugation. Nuclear Fraction A contained essentially homogeneous vesicular plasma membrane element's with little other cell components (Fig. 3A). Fract,ion B cont'ained mainly mitochondria w&h some plasma membranes and microsomes, while the sedimented Fraction C cont'ained various cell component,8 including nuclei (elcc-tron micrographs are not shown). Fraction A from the microsomal fraction (Fig. 3L3) contained predominantly plasma menrbranes and Golgi with some unidentifiable structures. Fraction 13 (Fig.  3C) contained primarily small veyicles with some plasma menbranes, microsomes, and unidentifiable structures. Fraction C (Fig. 30) was predominantly smooth and rough endoplasmic reticulum. Thus, the findings with electron microscopy are consistent with the profile of enzyme markers and indicate that particulate guanylate cyclaic of rat liver is about cquall3-distributed between plasma membranes and microsomes. Further fractionation and characterization of the microsomal Fractions A and B are currently in progress to determine whether guanylatr ryclase is located in the components other than plasma membranes and microsomcs such as Golgi.
Soluble and particulate guanylatr cyclase separated with tentrifugation at 105,000 x g from homogenates of heart (1) and lung (4)   Liver preparations were prepared with discontinuous sucrose indicated. The soluble fraction was obtained from a supernatant gradients as described. Tubes containing the greatest activities in fraction of a rat liver homogenate after centrifugation at 78,900 X nuclear Fraction A and microsomal Fractions B and C were used g for 90 min. The free Mn2+ concentration of 3 rnM represents the in this experiment. All particulate activities were determined after cation concentration in excess of that of GTP and ATP. The high pretreatment with 1% Triton X-100 and with 1 mM GTP as despecific activity of particulate fractions is due to the activation scribed under "Materials and Methods." Other conditions are as and solubilixation of the enzyme by Triton X-100 (1). therefore, examined the properties of the fractions prepared with discontinuous sucrose gradients of rat liver. Treatment with 1% Triton X-100 at 4" for 60 min prior to assay increased the activity of guanylate cyclase in all of the particulate fractions from sucrose gradients 3-to IO-fold (not shown). The activity of the supernatant fraction of liver homogenate was increased only 50 to 100%. Table III summarizes the effects of several divalent cations on guanylate cyclase activity in soluble and purified particulate fractions. At a concentration of 3 rnhf, Mn2+ was much more effective than Ca2+ or Mg2f as a sole cation with all preparations.
When the concentration of Mn2f was lowered to 0.5 mM the activity of plasma membranes and microsomes decreased less than the soluble enzyme. This indicated that the soluble enzyme is more dependent upon free Mn2+ than the particle-bound enzymes. Chrisman et al. (4) have reported similar observations with preparations from rat lung. An important finding was the stimulation of guanylate cyclase activity from all fractions by Mg2f in the presence of low concentrations of Mn2+ (Table III). This is in contrast to previous reports of several laboratories that showed no effect of Mgz+ in the presence of Mn2+ (8,9,11). We have also found that MgClz enhanced the activity of guanylatc cyclase in soluble and particulate preparations from rat heart when MnClz is present. The effects of Ca2+ were strikingly different with each preparation (Table III and Fig. 4). Calcium ion inhibited the activity from fractions containing plasma membranes (nuclear Fraction A, microsomal Fraction A, and microsomal Fraction B), had little or no effect on the activity of microsomes (microsomal Fraction C), and markedly stimulated the soluble activity. The effects of calcium were qualitatively similar with or without pretreatment of enzyme preparations with Triton X-100. The stimulatory or inhibitory effects of calcium ion are also unaltered with Triton X-100 pretreatment of heart preparations (1). ATP inhibits crude preparations of guanylate cyclase from a number of tissues (1, 4-10). The inhibition by ATP was observed with all fractions from liver preparations but was somewhat greater with the soluble fraction (Table III). We previously reported that the soluble enzyme from rat heart was also more sensitive to ATP inhibition than the crude particulate enzyme (1).
Double reciprocal plots of velocity versus GTP concentration were linear with the soluble fraction and curved with the particulate fractions (not shown). Hill coefficients for GTP were 1.60 and 1.58 for the enzymes from plasma membranes (nuclear Fraction A) and microsomes (microsomal Fraction C), respectively, suggesting two or more interactive sites for GTP (Fig. 5). The Hill coefficient for the soluble preparation was 1.02 indicating a single site for GTP. The apparent K, for GTP for the soluble enzyme was calculated to be 35 PM from double reciprocal plots (not shown). With enzymes from plasma membranes and microsomes the concentration of GTP for half-maximal activity was 0.2 mM. The Hill coefficients and apparent K,s are similar to the values reported previously for the soluble and particulate activities from rat heart (I), lung (4), spleen (4), and liver (10).

DISCUSSIOli
The present study demonstrates that 80% of the guanylate cyclase activity in rat liver homogenates is associated with the soluble fraction. This value is similar to previous reports (l-3, 5, 7). The particulate guanylate cyclase activity is located in both purified plasma membranes and microsomes that were prepared by the method of Touster et al. (14). The enrichment of guanylate cyclase in plasma membranes paralleled that of 5'nucleotidase and adenylate cyclase, i.e. the recoveries and relative specific activities of the three enzymes were similar in Fraction A derived from the crude nuclear fraction (Table II). With electron microscopy this fraction appeared to be a homogeneous preparation of plasma membranes (Fig. 3A). On the other hand, the recovery of guanylate cyclase from crude microsomal fractions was similar to that of glucose-6-phosphatase in Fraction C, and it did not parallel that of 5'-nucleotidase and adenylate cyclase (Table II) strated that it was essentially homogeneous smooth and rough endoplasmic reticulum (Fig. 30). The recoveries and relative specific activities of guanylate cyclase, 5'-nucleotidase, and adenylate cyclase were similar in Fraction A from the crude microsomal pellet (Table II). From homogenates 5'-nucleotidase and adenylate cyclase were purified 2% and 11-fold, respectively, in this fraction. These values are comparable to previous reports (14, 29). Touster et al. (14) characterized this fraction as an enriched preparation of plasma membranes with the use of other criteria. Thus, we can conclude that guanylate cyclase in crude microsomal fractions is present in both plasma membranes and microsomes. However, the electron micrographs of Fraction A from the crude microsomal fraction did not demonstrate homogeneous plasma membranes (Fig.  3B) ; significant quantities of Golgi were present as characterized by cisternae and vesicles containing dense particles that are presumably very low density lipoproteins. Since the specific activities of guanylate cyclase, adenylate cyclase, and 5'-nucleotidase were similar in Fraction A from either crude nuclear or crude microsomal fractions (Table II), we can conclude that these three enzymes are located in other cell components such as Golgi. Activities were determined with 4 mM MnC& and various concentrations of GTP. Activities of the nuclear A (2.9 JL~ of protein/tube) and microsomal C (47 rg of protein/tubej fractions from discontinuous sucrose gradients were determined after treatment with 0.5% Triton X-100. The soluble fraction (8 pg of protein/tube) was'purified with Bio-Gel P-300 column chromatography as described previously (1 Fraction B from the crude microsomal fraction contains large amounts of guanylate cyclase, 5'-nucleotidase, and adenylate cyclase. Since little of the glucose-6-phosphatase activity was obtained in this fraction, it seems unlikely that this can be attributed to microsomes in the preparation. The electron micrographs demonstrated primarily small vesicles with some plasma membranes and microsomes. Additional studies to further characterize this fraction are in progress in this laboratory. It has been postulated that the endoplasmic reticulum provides precursors for Golgi that in turn provide precursors or determinants of the cell membrane and lysosomes (33). If indeed this is true, nucleomicrosomal enzymes such as particulate guanylate cyclase, adenylate cyclase (28)) 5'-nucleotidase (26)) sialidase (34), and others might be expected to be found in all of these structures. Some reports have described Y-nucleotidase in microsomes (21,27) and Golgi (32). Entman et al. (35) and Katz et al. (36) reported some adenylate cyclase activity in sarcoplasmic reticulum of heart. Adenylate cyclase has also been re-ported in Golgi and plasma membranes from porcine pituitary (37). White (38) recently reported guanylate cyclase activity in rat heart sarcoplasmic reticulum. Some enzymes such as galactosyltransferase, glycolipid glucosyltransferase, and sulfotransferase are reported in Golgi and microsomes but not plasma membranes (39-41).
The properties of soluble and particulate guanylate cyclase from rat liver were similar to preparations from several other tissues (1, 4-11). As with other tissues the properties of the soluble and particulate enzyme were different. Calcium ion stimulated the soluble activity, inhibited the activity in plasma membranes, and had no effect on the activity in microsomes (Table III, Fig. 4). The effect of Ca2f has been the only difference that we have observed to date in the plasma membrane and microsomal enzyme. Mn2+ was much more active than Mg2+ or Ca2+ as the sole cation. However, in contrast to reports from other laboratories working with other tissues (8, 9, II), Mg2+ effectively stimulates all guanylate cyclase activities from rat liver preparations when low concentrations of Mn2+ were present (Table III). With the low concentrations of Mnyf normally present in tissues (42), the stimulatory effect of Mg2+ may be important in regulating cyclic GMP synthesis and accumulation.
Therefore, while most of the guanylate cyclase in liver is soluble, significant quantities are associated with plasma membranes and microsomes. The three or perhaps more subcellular locations of the enzyme raise a number of questions about their physiological significance and role in cyclic GMP synthesis. In other experiments we have found increased particulate guanylate cyclase activity and decreased soluble enzyme activity in regenerating rat liver, fetal rat liver, and hepatomas (43). These observations suggest that increased particulate enzyme is associated with liver growth and are of interest in view of the recent reports describing stimulatory effects of cyclic GMP on cell culture proliferation (44)(45)(46).