Association of integrated metabolic pathways with membranes

Green, D.E.; Mürer, Erik H.; Hultin, Herbert O.; Richardson, Stephen H.; Salmon, Bernard J.; Brierley, Gerald P.; Baum, Harold

doi:10.1016/0003-9861(65)90107-4

Cited by 221 publications

(34 citation statements)

References 15 publications

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“…Our results cannot be conclusive in this respect. However, it seems clear that the adherence of hexokinase activity to the plasma membrane as reported by some authors [21,22] can be excluded. A minor fraction of type III hexokinase is present too in fresh preparations of erythrocyte hexokinase [4].…”

Section: Discussionmentioning

confidence: 83%

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

Rijksen

Staal

Beks

et al. 1982

Biochimica et Biophysica Acta (BBA) - General Subjects

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The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP: l>hexose-6-phosphotransferase, EC 2.7.1.1) were studied in erythrocytes, blood platelets, lymphocytes and granulocytes. Soluble and particulate fractions were separated by a rapid density centrifugation method after controlled digitonin-induced cell lysis. In lymphocytes and platelets the major part of total activity was particle-bound (78 and 88%, respectively). In granulocytes and erythrocytes most of the hexokinase activity was found in the cytosol. All cell types, except granulocytes, contain mainly the type I isozyme. Platelets contain only type I hexokinase, while in lyphocytes a minor amount of type III is present in the soluble fraction (less than 10% of total activity). The major constituent of granulocytes is type III hexokinase (70-80% of total activity), the remaining 20-30% is type I hexokinase. Erythrocytes contain a multibanded type I hexokinase. The substrate affinities of the type I hexokinase do not differ significantly between the different cell types or between soluble, bound and solubilized fractions. Only soluble hexokinase from iymphocytes shows a slightly decreased K m apparent for glucose (P < 0.05).

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Section: Discussionmentioning

confidence: 83%

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

Rijksen

Staal

Beks

et al. 1982

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…The most frequently offered explanation for assembly of any enzyme complex argues that the efficiency of a pathway should increase with the channeling of substrates between sequential enzymes in a pathway (32). In this regard, it should be noted that there is mounting evidence that the glycolytic machinery in the human RBC can compartmentalize ATP, allowing its direct consumption by ion pumps without release into the cytoplasm (22)(23)(24).…”

Section: Discussionmentioning

confidence: 99%

Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane

Campanella

Chu

Low

2005

Proc. Natl. Acad. Sci. U.S.A.

356

327

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To characterize the location of glycolytic enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1-12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.GAPDH ͉ band 3 ͉ AE1 ͉ metabolon ͉ compartmentalization

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“…Association of cytosolic enzymes with the cytoskeleton was shown with members of the glycolytic pathway (18)(19)(20). Our report that CHS (a soluble enzyme) is associated with ER membranes in the plant cells is consistent with our model for the existence of a consecutively assembled membrane-associated enzyme complex in phenylpropanoid and flavonoid metabolism (3).…”

Section: Discussionmentioning

confidence: 99%

Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes.

Hrazdina

Zobel

Hoch

1987

Proc. Natl. Acad. Sci. U.S.A.

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Chalcone synthase [naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), E.C. 2.3.1.74], the key enzyme of flavonoid pathways that was believed to be soluble, has been localized on ribosome-bearing endoplasmic reticulum membranes in the epidermis of buckwheat (Fagopyrum esculentum M.) hypocotyls. Enzyme activity measurement and immunoblots of buckwheat hypocotyl homogenates that were fractionated on linear sucrose density gradients and developed with a specific chalcone synthase antibody and a 20-nm ImmunoGold coijugate showed the presence of chalcone synthase in fractions enriched in endoplasmic reticulum membranes. The presence of chalcone synthase on these membranes was not caused by nonspecific adsorption or entrapment of proteins. Immunocytochemical investigations with both a 5-nm and a 20-nm ImmunoGold conjugate showed that chalcone synthase was associated with the cytoplasmic face of rough (ribosome bearing) endoplasmic reticulum membranes. Plasma membrane, nucleus, plastids, mitochondria, golgi, and the tonoplast were not labeled. These data are consistent with our earlier described model suggesting that the synthesis of phenylpropanoids and flavonoids takes place partially or fully on membrane-associated enzyme complexes.Flavonoid compounds are produced by most, if not all, higher plants and are synthesized from p-coumaryl-CoA and malonyl-CoA by the enzyme chalcone synthase [CHS; naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), EC 2.3.1.74]. Cell fractionation experiments showed that the majority of the activity of this enzyme was "soluble," with small but significant portions associated with a membrane fraction (1). Ultracentrifugation (2) and gel-filtration experiments (3) suggested that CHS is associated with the endoplasmic reticulum (ER) membranes. Convincing evidence exists that another soluble enzyme of this pathway, phenylalanine ammonia lyase (E.C. 4.3.1.5) is at least in part membrane associated (4).Detection of enzyme activities in the soluble cytoplasmic fraction after cell fractionation is not a convincing argument for subcellular localization in vivo. Most homogenization procedures destroy cellular organization, and most suspension media that are used during cell fractionations are not physiological in nature. It is becoming increasingly clear that many so-called cytosolic enzymes interact with each other and the structural components of the cell, and the enzyme activities are modified by these interactions (5). Thus, the term "soluble enzyme" should not be taken as indicating that the catalytically active compartment is a true solution. Rather, it should be regarded as an operational definition, referring more to the result of homogenization and centrifugation procedures in subcellular fractionation than to any established in vivo characteristic.Our previous studies on localizing weakly associated enzymes or ER membranes by activity alone were greatly hampered by inactivation of enzymes during the isolati...

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Association of integrated metabolic pathways with membranes

Cited by 221 publications

References 15 publications

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane

Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes.

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