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 phos- phatase 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 phospha- tase 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 en- zyme 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 en- zyme . 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 homoge- nizer 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