Association of Glycogen Synthase Phosphatase And

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ASSOCIATION OF GLYCOGEN SYNTHASE PHOSPHATASE AND PHOSPHORYLASE PHOSPHATASE ACTIVITIES WITH MEMBRANES OF HEPATIC SMOOTH ENDOPLASMIC RETICULUM Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021

R . N . MARGOLIS, R . R . CARDELL, and R . T . CURNOW

From the Departments of Anatomy, Internal Medicine, and Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

ABSTRACT A detailed investigation was conducted to determine the precise subcellular localization ofthe rate-limiting enzymes of hepatic glycogen metabolism (glycogen synthase and phosphorylase) and their regulatory enzymes (synthase phosphatase and phosphorylase phosphatase) . Rat liver was homogenized and fractionated to produce soluble, rough and smooth microsomal fractions . Enzyme assays of the fractions were performed, and the results showed that glycogen synthase and phosphorylase were located in the soluble fraction of the livers . Synthase phosphatase and phosphorylase phosphatase activities were also present in soluble fractions, but were clearly identified in both rough and smooth microsomal fractions . It is suggested that the location of smooth endoplasmic reticulum (SER) within the cytosome forms a microenvironment within hepatocytes that establishes conditions necessary for glycogen synthesis (and degradation) . Thus the location of SER in the cell determines regions of the hepatocyte that are rich in glycogen particles . Furthermore, the demonstration of the association of synthase phosphatase and phosphorylase phosphatase with membranes of SER may account for the close morphological association of SER with glycogen particles (i .e ., disposition of SER membranes brings the membrane-bound regulatory enzymes in close contact with their substrates) .

KEY WORDS smooth endoplasmic reticulum less clear . Most workers have noted that glucose- glycogen synthesis - synthase phosphatase 6-phosphatase is found in smooth microsomas (9, phosphorylase phosphatase 19), and the cytochemical localization of the enzyme to rough and smooth endoplasmic reticulum Early studies on the fine structure of hepatocytes (RER and SER) has been achieved (19, 31) . These showed a close association of smooth endoplasmic observations have led to the conclusion that SER reticulum (SER) and glycogen particles (4, 12, 25) . is involved in glycogen breakdown and/or glucose Almost all later investigators have confirmed this release from the cell. However, studies involving close morphological association of SER and gly- animals maintained on a controlled feeding sched- cogen, but the functional implications have been ule (1, 2) have suggested that SER is associated

348 J . CELL. BIOLOGY © The Rockefeller University Press - 0021-9525/79/11/0348/09 $1 .00 Volume 83 November 1979 348-356

with glycogen particles during glycogen deposi- basis of this information and the morphological tion . In addition, SER was found closely associ- evidence cited above, Dallner and Ernster (10) ated with glycogen particles in hepatocytes of ad- suggested the possibility that synthase phosphatase renalectomized rats injected with a glucocorticoid is associated with membranes of the SER . Cardell (5, 23) . Under these conditions, it is clear that the (6) also regarded this as a likely possibility . hepatocytes are actively depositing glycogen rather In this paper, we report careful fractionation than breaking down the carbohydrate . Thus, some studies in which highly purified smooth and rough morphological evidence suggests a role for SER in microsomal fractions were prepared and assayed the synthesis of hepatic glycogen (6) . for the rate-limiting enzymes of glycogen metab- Previous attempts to relate enzymes involved in olism and for the converting enzymes (synthase hepatic glycogen synthesis to the SER have been phosphatase and phosphorylase phosphatase) . largely unsuccessful . However, much information Our results clearly show significant quantities of has been accumulated on the biochemical mech- both synthase phosphatase and phosphorylase Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021 anisms for hepatic glycogen synthesis . It has been phosphatase in the microsomal fractions of livers established that the rate-limiting enzymes of gly- from fasted rats . In subsequent and more detailed cogen synthesis and degradation are glycogen syn- publications, we will report our findings on the thase and glycogen phosphorylase, respectively properties of the membrane-bound enzymes and (14, 27) . These enzymes exist in physiologically their response to various hormonal and dietary active and inactive forms with rapid enzymic in- manipulations of the experimental animals . terconversion between the two forms of each enzyme . The chemical nature of the interconversion MATERIALS AND METHODS reactions of these enzymes involves phosphoryla- Animals tion by specific kinases (16, 17) and dephospho- Adult, male Wistar rats (200-250 g) by phosphatases which were used in all experi- rylation may be specific or ments . Rats were allowed ad lib . access to food and water, but nonspecific (8, 16, 17) . The physiologically active were fasted for 24 h before sacrifice . All rats were maintained on form of glycogen synthase is the dephosphorylated a 12 :12 h light-dark cycle . or I form, whereas the phosphorylated or D form is inactive under physiological conditions (16-18) . Electron Microscopy Conversely, the physiologically active form of gly- Animals were decapitated, a portion of the left lateral lobe of cogen phosphorylase is phosphophosphorylase (a each liver was rapidly removed, and the sample was placed in a drop of in form) with the dephospho- form of the enzyme (b 3"%, glutaraldehyde 0.1 M cacodylate buffer (pH 7 .3) . The tissue was cut into small pieces -I mm' in size and placed form) being inactive (16-18) . in a vial containing the glutaraldehyde fixative . After 2 h of Luck (2l) and several subsequent investigators fixation at room temperature, the tissue was rinsed in cacodylate (15, 18) fractionated liver cells and studied the buffer (0 .1 M, with 10`;i sucrose) and postfixed in I'ii osmium distribution of glycogen synthase and phosphoryl- tetroxide (in 0.1 M phosphate buffer) . The tissue was then . These workers dehydrated in a graded series of alcohol and embedded in Epon ase concluded that the enzymes (22) . Ultrathin sections were stained with uranyl acetate and lead are either associated with glycogen particles or citrate (26. 32) and examined in a Philips EM-300 electron found in the soluble component of the cell . No microscope . reported evidence exists for the localization of these enzymes in either SER or RER. However, it Subcellular Fractionation should be noted that Hizukuri and Larner (l5) Livers from 24-h fasted rats' were excised, blotted dry, provided an important early finding when they weighed, and placed in cold 0.25 M sucrose . Homogenization demonstrated a "converting" factor in a glycogen- was carried out with a motor-driven Potter-Elvejhem homogenizer in a cold room (0°-4°C) . A 20'7, (wt/vol) homogenate was free fraction that sedimented after high-speed cen- obtained by making three up-and-down passes of the pestle (900 trifugation . It was suggested that this fraction was rpm) in the homogenization vessel. Homogenates were centri- possibly of microsomal origin ; however, to our fuged twice at 10.000g for 20 min in a Beckman J-21C (Beckman knowledge, no further attempts were made to clar- Instruments. Inc ., Spinco Div., Palo Alto, Calif.) preparative centrifuge. The resultant postmitochondrial ify this point . This fraction catalyzed the conver- supernate (PMS) was saved, and the pellet containing nuclei, plasma membrane, mi- sion of glycogen synthase from inactive to active tochondria . and other cellular debris was discarded . form in rat liver. Subsequent investigations showed that this enzymatic reaction was a de- ' In these initial experiments, fasted animals were used phosphorylation of glycogen synthase caused by to avoid contamination of the fractions with glycogen the enzyme synthase phosphatase (29) . On the particles .

MARGOLIS, CARDELL. AND CURNow Localization of Hepatic Glvcogen Enzymes 349

Smooth and rough microsomes were prepared by the Dallner I U representing I U of phosphorylase a converted to phospho- et al . (9, 10) procedure . Briefly, the PMS was made 15 mM Cs' rylase b per minute . with I M stock solution of CsCl . The PMS plus 15 mM Cs' was Separate portions of each subcellular fraction were also as- layered over 15 ml of 1 .3 M sucrose plus 15 mM Cs' in an SW27 sayed for glycogen synthase activity and phosphorylase activity . centrifuge tube (Beckman Instruments) . Centrifugation in an L5- a s previously described (7, 13, 30) . 50 (Beckman Instruments) ultracentrifuge for 4 h at 105,000g at Significance of differences between means was determined by 4°C produced a pellet containing rough microsomes beneath the Student's ( test . Activities are presented as means ± standard 1 .3 M sucrose and a band containing smooth microsomes at the errors (SEM) of numbers of determinations . 0.25-1 .3 M sucrose interface . The supernate above the band was drawn off (soluble fraction) and saved on ice . The band was RESULTS drawn oft and diluted with distilled water, and the pellet was resuspended in 0.25 M sucrose by gentle homogenization in a Hepatocytes from 24-h fasted rats contained gly- glass homogenizer. Both subfractions were centrifuged in a Ti50 cogen particles restricted to distinct regions of the rotor at 225,000 g for I h . Final pellets were resuspended in 0.25 cytosome (Fig. l) . Within the glycogen regions of M sucrose . the cell, numerous tubules and vesicles of SER Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021 Chemical and Enzymatic Determinations were closely associated with the particles of gly- Protein was determined by the method of Lowry et al . (20) . cogen (inset, Fig . I) . It is clear from such micro- Glycogen was determined by extraction of tissue portions in 30% graphs that the distribution of SER in the cyto- KOH and precipitation of glycogen with 66% ethanol. Glycogen some of hepatocytes is related to the location of was estimated by hydrolysis via the phenol-sulfuric acid method glycogen within the cells . (11) . Glycogen synthase phosphatase activity in the various frac- After homogenization and fractionation of rat tions was determined in a two-stage incubation assay . The first liver, rough and smooth microsomal subfractions incubation involved the conversion of purified glycogen synthase were obtained . Electron microscopy of ultrathin from the D to the I form . This stage was initiated by adding 8 sections of the pellets from these fractions showed pl of the subcellular fraction to a mixture of 5 td of a solution containing 1 .2 U- of muscle glycogen synthase D, 10 mM Tris that the rough microsomes were isolated predom- (pH 7.8).I mM EDTA, 0.5 mM EGTA. 0.5 mM phenymethyl- inantly as vesicles containing ribosomes bound to sulfurylurea fluoride, 10 mM mercaptoethanol, 5% glycerol . and the outer, cytoplasmic surface (Fig . 2), and hence 8 mg/ml glycogen . To this was added 4 AI of H,O or 4AI of 42.5 were derived from the RER. The smooth micro- mM MgCl, . Incubation for varying lengths of time at 30'C was somal fraction appeared as smooth-surfaced vesi- carried out . with the phosphatase reaction stopped by adding 20 Itl of ice-cold 400 mM KF and placing the assay tube on ice . The cles, with occasional flattened saccules and sheets second stage of the phosphatase assay involved determining the of membranes (Fig . 3) . We regard this fraction as synthase 1 activity generated in the first stage by the filter paper containing mainly elements of SER; however, con- method of Thomas et al . (30) . The synthase phosphatase reaction tamination by other cellular organelles (Golgi ap- was linear for all fractions over 20 min . Glycogen synthase D paratus, plasma was purified by the method of Takeda et al . (28) . Synthase membrane, etc .) may represent a phosphatase is presented as units per gram protein in the subcel- minor component (9, 23) . Further support for this lular fraction, with I U of synthase phosphatase equal to I U of conclusion was provided by determinations of synthase D converted to synthase 1 per minute. glucose-6-phosphatase specific activity (data not Glycogen phosphorylase phosphatase activity in identical shown) which showed that the smooth microsomal fractions was also determined by a two-stage assay . Enzymatic fraction is rich in this enzyme, and hence contains conversion of phosphorylase a (Sigma Chemical Co ., St . Louis, Mo .) to phosphorylase b was determined by incubation for predominantly membranes of SER . In addition, varying lengths of time at 30'C of hepatic subcellular fractions analysis of RNA content revealed that 95% of the in buffer containing 4 U/ml"phosphorylase a, 20 mM glycylgly- RNA is localized in rough microsomes (data not 7 .8), MgCl., cine (pH 10 mM 10 mM caffeine, 0 .1!%7 albumin, shown) . and 10!'r glycerol . This reaction was stopped by adding ice-cold KF in MES (pH 6.1) to give final concentrations of 66 mM and We measured the activity of glycogen synthase 16 mM, respectively, with the tubes subsequently placed on ice . in four subcellular fractions : PMS, soluble, rough Phosphorylase a activity was then determined on these samples microsomes, and smooth microsomes . The enzyme by the filter paper method of Gilboe et al (13) . Phosphorylase was found in both PMS and soluble fractions, phosphatase activity is presented in units per gram protein. with whereas no enzyme activity beyond trace amounts was detected in microsomal subfractions (Fig . 4) . -' Glycogen synthase D is expressed as units (micromoles A similar distribution of glycogen phosphorylase of glucose incorporated into glycogen from uridine diphosphate glucose per minute per milligram protein in was observed (Fig. 5) . Thus, specific activity of the presence of 10 mM glucose-6-phosphate) . these rate-limiting enzymes was detected in PMS ` Phosphorylase a is also expressed as units (micromoles and soluble fractions of the livers, but not in of glucose incorporated into glycogen from glucose-I- significant quantities in the rough and smooth phosphate per minute per milligram protein) . microsomal fractions .

350 THE JOURNAL OF CELL BIOLOGY " VOLUME 83, 1979 Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021

FIGURE 1 Low magnification (x 10,000) electron micrograph of centrilobular hepatocyte from overnight-fasted normal rat. Note glycogen area (arrows) containing numerous tubules and vesicles of SER and glycogen particles . Numerous areas of cytoplasm rich in RER are obvious . Mitochondria (M) and nucleus (N) are also identified. A higher magnification (x 36,000) of a glycogen region from a centrilobular hepatocyte is displayed in the inset . Note close proximity of glycogen (G/) to SER . Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021

FIGURE 2 Electron micrograph of a section through the rough microsomal pellet showing ribosomes (R) on the outer (cytoplasmic) surface of vesicles . x 54,500.

FIGURE 3 Electron micrograph of section through the smooth microsomal pellet . Only occasional vesicles have ribosomes (R) attached to membranes . x 54,500 .

160 specific activity in both rough (42.4 ± 4.5 U/g) 150 and smooth (31 .1 ± 6.2 U/g) microsomes . It is IjSEM 140 noteworthy that a significant amount of enzyme p 130 was demonstrated in the soluble fraction (9 .2 ± X 120 2.2 U/g) as well . 110 200 loo 190 . 90 d 180 O 80 n. 170 70 oI 160 E 60 50 O_ 150 u 140 40 X 130 30 I ± SEM 20 120 10

PMS Soluble SER RER FIGURE 4 Glycogen synthase activity in hepatic sub- E 80 w cellular fractions from overnight-fasted normal rats . En 70 zyme-specific activity is presented as micromoes C product 60 formed per milligram protein per minute . Each point 50 represents the mean of four determinations ± SEM. See 40 text for details of assay procedure. PMS, postmitochondrial supernate ; Soluble, postmicrosomal supernate ; 30 SER, smooth microsomes ; RER, rough microsomes . 20 10 In contrast, phosphorylase phosphatase activity was measured in eight subfractions, and was PMS Soluble SER RER clearly present in hepatic microsomal membrane FIGURE 5 Glycogen phosphorylase activity in hepatic fractions obtained from the livers of fasted rats subcellular fractions from overnight-fasted normal rats. (Table I) . The enzyme was detected with highest See Fig . 4 and text for details .

352 THE JOURNAL OF CELL BIOLOGY " VOLUME 83, 1979

The protein concentrations, total protein, phos- mogenization procedure employed in these exper- phorylase phosphatase specific activity, total phos- iments to minimize contamination of ER mem- phorylase phosphatase activity, and enzyme recov- brane fractions with other cellular membranes re- ery in each fraction of a representative experiment sults in an appreciable loss ofphosphorylase phos- are presented in Table 1 . As can be seen, a signif- phatase activity in the initial centrifugation steps, icant amount of both total protein and phospho- it is also clear that the majority of this activity rylase phosphatase activity were lost in the first sediments in nearly equal amounts in the SER and two 10,000 g centrifugations (65 and 48%, respec- RER fractions. tively) . However, and more importantly, the ma- Synthase phosphatase was also localized in the jority of the phosphorylase phosphatase activity hepatic microsomal fractions where 1 .29 ± 0.11 recovered (114% of activity applied) from the dis- U/g of enzyme activity was observed in rough continuous sucrose gradient centrifugation step microsomes and 5.09 ± 0.45 U/g in smooth mi- was associated with the SER and RER fractions . crosomes (Table 11) . The soluble fraction dis- Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021 Thus, although it is clear that the very mild ho- played 2.70 ± 0.22 U/g enzyme specific activity .

TABLE l Subcellular Distribution of Phosphorylase Phosphaiase Activity in Liver

Phosphoryl- Phosphoryl- Protein con- ase phospha- ase phospha- Enzyme re- Supernate 11 centration Total protein tase activity tase activity covery activity

mglml g U lg. U 20' ( homogenate (10,000 g for 20 min) Supernate 17 .0 0 .493 13 .0 6 .41 49 Pellet 35 .5 0.553 12 .0 6 .63 51 Supernate 1 (10,000 g for 20 min) Supernate 11 15 .3 0,367 18 .6 6 .83 52 Pellet 12.6 0.126 15 .2 1 .92 15 Supernate II (105,000 g for 2 h on sucrose gradient)* Supernate (soluble) 5 .2 0.186 9 .2 1 .71 13 22 SER 3 .9 0 .070 31 .1 2 .17 16 28 RER 3 .5 0.063 42 .4 2 .66 20 34 1 .3 M sucrose 3 .0 0 .053 23 .2 1 .23 9 16 * See text for details .

TABLE 11 Subcellular Distribution of Glycogen Synthase Phosphatase Activity in Liver

Synthase phosphatase activity

Ulg Ulg UU Enzyme recovery Supernate Il activity

20% homogenate (10,000 g for 20 min) Supernate I 4.37 2.62 2.15 1 .29 65 69 Pellet 1 .36 1 .02 0 .75 0.56 35 31 Supernate I (10,000 g for 20 min) Supernate 11 3 .79 1 .67 1 .39 0.61 48 33 Pellet 4 .20 3.45 0 .51 0.43 18 15 Supernate II (105,000 gfor 2 hon sucrose gradient) Supemate (soluble) 4 .42 2 .70 0 .82 0 .50 28 27 66 42 SER 3 .07 5 .09 0 .21 0 .36 7 19 17 31 RER 0 .77 1 .29 0 .05 0 .08 2 4 4 7 1 .3 M sucrose 3 .13 4 .38 0 .16 0 .23 6 12 13 20 See text for further details . * Activities were determined in the presence (+) or the absence (-) of 10 mM Mg'= .

MARGOLIS, CARDELL, AND CURNow Localization of Hepatic Glycogen Enzymes 353 Several reports exist (3, 14, 17) of effects of diva- These findings assume particular importance be- lent cations on phosphoprotein phosphatase activ- cause these enzymes serve as potential regulators ities in crude extracts made from muscle and liver. of hepatic glycogen synthesis and breakdown . Consequently, we investigated the effects of diva- Their localization in ER membranes, particularly lent cations on our hepatic subcellular fractions the SER, may partially explain the close morpho- and found that magnesium had dramatic and op- logical association between glycogen particles and posite effects on enzyme-specific activity, depend- SER membranes during glycogenesis and glyco- ing on whether the enzyme was soluble or mem- genolysis in liver . This is especially true for syn- brane associated . Thus, when Mg*- was present, thase phosphatase activity which is present almost enzyme activity was decreased to 0.77 ± 0.22 U/g exclusively in these membranes . in rough and 3.07 ± 0.56 U/g in smooth micro- Previous investigations into the subcellular lo- somal fractions . In marked contrast, in the pres- calization of these enzymes (l5, 29) indicated that ence of Mg+', enzyme activity increased in the a small amount of phosphoprotein phosphatase Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021 soluble (4 .42 ± 0.50 U/g) fractions . Moreover, (i .e., "converting") activity resided in a fraction when synthase phosphatase was solubilized by that sedimented after high-speed centrifugation . high salt treatment, the solubilized enzyme re- Hizukuri and Larner (15) suggested that this frac- tained its sensitivity to Mg12 and thus could still tion was of microsomal origin, but no steps were be distinguished from the soluble form of the taken to substantiate this initial finding . Subse- enzyme. In preliminary studies, we have found quent reports have dealt mainly with characteri- that the K;, of soluble enzyme for Mg12 is 2 mM, zation and purification of soluble or glycogen- whereas the K; of the SER enzyme is 1 .5 mM .' associated phosphoprotein phosphatases from The quantitative distribution of glycogen syn- crude homogenates of either skeletal muscle or thase phosphatase activity measured in the pres- livers (7, 13, 16, 17) from rats and other laboratory in ence and absence of 10 M Mg12 in the same liver animals . fractions shown for phosphorylase phosphatase The methods of subcellular fractionation and activity in Table I is shown in Table 11 . Similar to enzyme assay reported in this paper have permit- phosphorylase phosphatase activity, -50% of the ted a clear determination of the precise localization synthase activity measured in the presence of mag- of synthase phosphatase and phosphorylase phos- nesium was lost during the initial low-speed cen- phatase in the hepatocyte . Accordingly, the pres- trifugation steps . In contrast, only one-third of the ence of synthase phosphatase and phosphorylase activity measured in the absence of magnesium phosphatase in the soluble fraction obtained from was recovered in the PMS . The recovery of syn- the hepatocyte confirms previously cited reports thase phosphatase activity applied to the discon- (7, 13, 16, l7) ; however, the localization of both tinuous sucrose gradient was 89% measured in the enzymes in the microsomal fraction and particu- presence of added 10 mM magnesium and 192% larly the smooth microsomal fraction is a new and measured in the absence of the cation . The distri- significant finding . Furthermore, our observation Mg12 bution of synthase phosphatase activity among the of a stimulation by of the soluble form of various fractions differed markedly from that of synthase phosphatase,in accordance with reports phosphorylase phosphatase in that nearly all of cited in the literature (17), is countered by the the former activity was associated with the SER, observed inhibition of membrane-bound synthase whereas the latter was distributed equally between phosphatase by Mg + ` . Indeed, the membrane- SER and RER. The data also demonstrate that bound enzyme exhibited higher specific activity in >50% of the synthase phosphatase activity in the the absence of Mg'`. Magnesium thus provides us PMS is associated with ER membranes . a probe for delineating between soluble and membrane-associated forms of synthase phosphatase . DISCUSSION Indeed, situations in which significant amounts of Mg12 The most significant observations reported in this enzyme are activated by may be indicative communication are that significant amounts of of the presence of large amounts of soluble enzyme synthase phosphatase and phosphorylase phos- as opposed to membrane-associated enzyme, as is phatase activities are present in ER membranes . the case in the PMS fraction. Because -32% ofthe intracellular Mg12 in liver is associated with ER ' Curnow, R . T ., and R . N . Margolis . Manuscript in membranes (24), it is possible to speculate that preparation . Mg`` could have an in vivo role in the regulation

354 THE JOURNAL OF CELL BIOLOGY " VOLUME 83, 1979

of this enzyme, perhaps with respect to binding of thesis . As glycogen synthesis progresses, accumu- the enzyme and/or controlling the activity once it lations of glycogen appear in the hepatocyte and is bound to the membrane . are identified in the electron microscope as "gly- The localization of both synthase phosphatase cogen-rich" areas of the cytosome . and phosphorylase phosphatase to rough and The evidence presented in this communication smooth microsomal fractions may provide the link suggests that these glycogen regions are rich both between the morphological observations of in- in converting enzymes (synthase phosphatase and creased SER during glycogen synthesis (2, 5), close phosphorylase phosphatase) and in the rate-timit- association of SER membranes and glycogen par- ing enzymes of glycogen synthesis and depletion ticles (2, 4, 5, 23), and the well-established bio- (glycogen synthase and glycogen phosphorylase) . chemical pathways of hepatic glycogen synthesis Obviously, much more work is required to fully (18, 27). In addition, preliminary investigations in characterize the hormonal, nutritional, and meta- our laboratories suggest that both membrane- bolic factors that regulate synthase phosphatase Downloaded from http://rupress.org/jcb/article-pdf/83/2/348/1074048/348.pdf by guest on 26 September 2021 bound forms of synthase phosphatase and phos- and phosphorylase phosphatase activation, syn- phorylase phosphatase are increased in amount thesis, and association with SER . However, we and/or activity in fed rats . Therefore, it is conceiv- feel that the results presented in this paper clearly able that during periods of glycogen deposition demonstrate a functional connection between SER both synthase phosphatase and phosphorylase and glycogen synthesis in the rat hepatocyte . phosphatase are activated, with the balance of enzymatic interconversion of glycogen synthase Grateful acknowledgement is made to Dr. Hisako Ku- and glycogen phosphorylase subsequently shifted kuchi for the purification of glycogen synthase D and to toward glycogen synthesis. Conversely, during pe- the technical assistance of Ms, Gwenn Clark . riods of glycogen depletion, both synthase phos- This work was supported by grants AM-1 1854, 21253, phatase and phosphorylase phosphatase are inac- 00477, and 20787 from the U.S . Public Health Service, tivated, with the balance then shifted toward gly- and in part by the University of Virginia Diabetes Re- search and Training Center (AM-22125) . cogen breakdown . In summary, we envision a situation in which Receivedfor publication 28 February 1979, and in revised SER forms a localized microenvironment within form 7 June 1979. certain areas of the hepatocyte and in which the regulatory enzymes synthase phosphatase and REFERENCES phosphorylase phosphatase are bound to the cytoplasmic surface of the SER . It is difficult at this I . BAncocK. M . B ., and R . R . CARDIA .L, JR. 1974 . Hepatic glycogen patterns in fasted and fed rats. Am. J . Anal . 140:299-338. time to reconcile the presence of phosphorylase 2 . BAR( OCK. M . B ., and R . R . CARDRm ., JR. 1975 . Fine structure of hepatocytes from fasted and fed rats . Am . J. Anal. 143 :399-438. phosphatase activities and, to a much lesser extent, 3 . Bt . acKMORE, P . F ., F . T . BRCMirv, 1 . L . M RKS. and 1 . H . Ex iON . of synthase phosphatase activities, in RER, as well, 1978 . Studies on o-adrenergic activation of hepatic glucose output. Relationship between it-adrenergic stimulation of calcium efflux and with the clear-cut finding of glycogen deposits activation of phosphurylase in isolated rat liver parenchymal cells . J. which are exclusively opposite elements of the Riot . Chem . 25 3 :4851 4858 . 4 . BRI'N1, C . . and K . R . PORrr:R. 1965 . The fine structure of the paren- SER. However, we feel that it is both the presence chymal cell of the normal rat liver . 1 . General observations . Am . J. of these enzymes on SER and the location of the Paihol . 46:691-755. 5 . CARDeLr. R . R . . 1R. 1974 . Action of metabolic hormones on the fine SER in the cytoplasm that are crucial factors . In structure of rat liver cells. III . Effects of adrenalectomy and adminis- either eventuality, it is the position of these en- tration of cortisone. Anal . Rec. 180;309-330 . 6. CARm it . R . R .. 1R . 1977 . 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356 THE JOURNAL OF CELL- BIOLOGY - VOLUME 83, 1979