Tracing Metabolic Fate of Mitochondrial Glycine Cleavage System Derived Formate In Vitro and In Vivo

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.

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Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation

Blood ◽

10.1182/blood.v63.5.1060.1060 ◽

1984 ◽

Vol 63 (5) ◽

pp. 1060-1066 ◽

Cited By ~ 15

Author(s):

M Miura ◽

CW Jackson ◽

SA Lyles

Keyword(s):

Bone Marrow ◽

Plasma Concentration ◽

Bone Marrow Cells ◽

Single Cells ◽

Rat Plasma ◽

Whole Body ◽

Growth Promoting ◽

Marrow Cells

Abstract To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

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Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation

Blood ◽

10.1182/blood.v63.5.1060.bloodjournal6351060 ◽

1984 ◽

Vol 63 (5) ◽

pp. 1060-1066 ◽

Cited By ~ 1

Author(s):

M Miura ◽

CW Jackson ◽

SA Lyles

Keyword(s):

Bone Marrow ◽

Plasma Concentration ◽

Bone Marrow Cells ◽

Single Cells ◽

Rat Plasma ◽

Whole Body ◽

Growth Promoting ◽

Marrow Cells

To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

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Megakaryocytes Exchange Significant Levels of Their Alpha-Granular PF4 with Their Environment

Blood ◽

10.1182/blood.v124.21.1432.1432 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1432-1432 ◽

Cited By ~ 3

Author(s):

Michele P Lambert ◽

Ronghua Meng ◽

Dawn Harper ◽

Liqing Xiao ◽

Michael S. Marks ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Whole Body ◽

Platelet Factor ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Background Staining ◽

The Media

Abstract Platelet factor 4 (PF4, CXCL4) is a major chemokine in megakaryocytes (megs). It is synthesized almost exclusively by megs during their development and may have important roles in regulating both hematopoietic stem cell and megakaryocyte proliferation. We now show that megs both release significant amounts of PF4 into their environment as well as take up PF4 into alpha granules. This PF4 is then available for release by thrombin activation. We examined PF4 recycling during megakaryopoiesis based on the observation that in vitro-cultured human meg hematopoietic precursors release significant amounts of PF4 into the media beginning after approximately 7 days of culture, when definitive megs begin to emerge. Using immunohistochemistry, we find that in vivo in murine bone marrow, human PF4 (hPF4) is released by hPF4 transgenic (hPF4+) megs during the steady-state, and this release is markedly accentuated 48 hours after sub-lethal 660 cGy whole body irradiation from an X-ray source to induce bone marrow injury. By comparison, animals without endogenous PF4 expression (Pf4-/-) showed only background staining. After irradiation, the levels of PF4 staining within the hPF4+ megs decreased with a concomitant increase in background staining suggesting that the stored PF4 was released into the bone marrow milieu. The increase in the PF4 staining in the intramedullary space was not due to released PF4 from entrapped platelets as similar changes were seen in untreated hPF4+ mice and in mice made thrombocytopenic by injection of antiCD41 antibody. We then asked whether the released PF4 could be taken back up by the megs and whether internalized PF4 could reach significant levels compared to endogenously synthesized PF4. We show that murine megs can take up significant levels of hPF4 so that peak hPF4 uptake at 24 hours (19±2 ng/10e6 cells) is equivalent to the amount of mouse (m) PF4 (30±1 ng/106 cells) natively present within the megs. Blocking antibodies to either PF4 itself or to lipoprotein receptor related protein 1 (LRP1) prevented PF4 uptake (53±17 IU/10e6 cells and 32±9 IU/10e6, respectively, vs 95±9 IU/10e6 cells, p <0.01, for either vs. no treatment), consistent with our previous report that LRP1 was necessary for PF4’s negative paracrine effect on megakaryopoiesis. The PF4 that was taken up by megs localizes at least in part to alpha granules, as evidenced by co-localization with P-selectin by immunofluorescence microscopy. Quantification showed a higher degree of colocalization between endogenous mPF4 and internalized hPF4 than between other alpha-granule markers, including vWF, P-selectin and internalized fibrinogen. Moreover like endogenous mPF4, the internalized PF4 can be re-released upon thrombin-induced meg activation. Finally, we asked whether the PF4 uptake was unusual and began by studying uptake of the related chemokine, platelet basic protein (PBP, CXCL7), another protein synthesized by megs and stored in alpha-granules. Unlike PF4, PBP was not internalized by megs as judged by immunohistochemistry or ELISA, indicating that the ability to be internalized and re-released is a relatively unique property of PF4. In summary, we demonstrate that PF4 - an important regulator of megakaryopoiesis and hematopoiesis - is released by megs in the intramedullary space at steady-state and even more so when stressed. Moreover, the released PF4 can be taken up into alpha-granules and stored for potential rerelease. Whether this complex cycle of PF4 in megs is unique to PF4 or applies to other alpha-granular proteins and whether it is necessary for the PF4 effect on hematopoiesis/ megakaryopoiesis needs further investigation Disclosures Xiao: ECRI Institute: Employment.

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Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611798114 ◽

2016 ◽

Vol 113 (52) ◽

pp. 15024-15029 ◽

Cited By ~ 5

Author(s):

Michael I. Carr ◽

Justine E. Roderick ◽

Hong Zhang ◽

Bruce A. Woda ◽

Michelle A. Kelliher ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Tumor Suppression ◽

Bone Marrow Failure ◽

Genotoxic Stress ◽

Whole Body ◽

Atm Kinase ◽

Radiation Induced

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2Y393F) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.

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Hematopoiesis in Regenerated Bone Marrow on the Hydroxyapatite Scaffold.

Blood ◽

10.1182/blood.v110.11.1419.1419 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1419-1419

Author(s):

Atsushi Fujita ◽

Makoto Migita ◽

Takahiro Ueda ◽

Yoshitaka Fukunaga ◽

Takashi Shimada

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Lentiviral Vector ◽

Wide Distribution ◽

Whole Body ◽

Hematopoietic Stem ◽

In Vivo Bioluminescence Imaging ◽

Hydroxyapatite Scaffold

Abstract Background: In bone marrow (BM), osteoblastic cells lining the endosteal surface are a key component of the niche to promote and regulate hematopoietic stem cells (HSCs). Anatomical architecture of BM could be regenerated by culturing BM derived stromal cells on the hydroxyapatite (HA) scaffold. In the present study, we examined whether the regenerated BM has the ability to support HSCs in vivo. Methods: Our study was designed as follows; BM stromal cells from C57/BL6 mice (Ly5.2) were cultured on the HA scaffold with numerous small pores for 3 days in vitro and the scaffold with attached cells was implanted subcutaneously onto the back of C57/BL6 recipient mice. 4.0x105 Lineage negative (Lin−) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered into the recipient mice after lethal irradiation. Eight weeks after transplantation, the scaffolds were removed from the first recipient mice and subcutaneously transplanted into the lethally irradiated second recipient mice. The mice also received fresh Ly5.2 BM cells for survival. Biodistribution and kinetics of Luc+ Ly5.1 cells were monitored by in vivo bioluminescence imaging and FACS. Results: In the secondary transplanted mice, Luc+ hematopoitic cells were detected in the scaffolds for at least 6 months after transplantation. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to whole body including the head, extremities, chest, and abdomen. The presence of Ly5.1 B and T lymphocytes in the circulation was confirmed by FACS analysis 5 months after secondary transplantation. Conclusions: The regenerated BM on the HA scaffold is capable of supporting HSCs in vivo suggesting that the functional niche is reconstituted. Hematopoiesis in the regenerated BM may have a significant impact for development of new therapeutic strategies for various hematopoietic diseases.

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CD200 Limits Monopoiesis and Monocyte Recruitment in Atherosclerosis

Circulation Research ◽

10.1161/circresaha.119.316062 ◽

2021 ◽

Author(s):

Christina Kassiteridi ◽

Jennifer E Cole ◽

Thibault Griseri ◽

Mika Falck-Hansen ◽

Michael E Goddard ◽

...

Keyword(s):

Bone Marrow ◽

Macrophage Activation ◽

Atherosclerotic Lesion ◽

Whole Body ◽

Limiting Factor ◽

Monocyte Recruitment ◽

Artery Disease

Rationale: Inflammation is a basic component of the pathogenesis of atherosclerosis. CD200 is an immune checkpoint known to control macrophage activation. CD200 recently emerged in the Framingham Heart Study and 2 other cohorts as being potentially relevant in CVD. The role of this pathway in CVD is unknown. Objective: We sought to examine the role of CD200 in atherosclerosis. Methods and Results: Using hypercholesterolemic ApoE-/- mice, we demonstrate that whole-body CD200 deficiency augments atherosclerotic lesion formation and vulnerability. Administration of a CD200-Fusion protein reduces neointima formation. Our data show that the CD200-CD200R pathway restrains activation of CD200R+ lesional macrophages, their production of CCR2 ligands, and monocyte recruitment in vitro and in vivo in an air pouch model. Loss of CD200 leads to an excessive accumulation of classical Ly6Chi monocytes and CCR2+ macrophages within the atherosclerotic aorta, as assessed by mass cytometry. Moreover, we uncover a previously uncharacterised effect of the CD200/CD200R pathway in limiting dysregulated monopoiesis and Ly6Chi monocytosis in hypercholesterolemic mice. Bone marrow chimera experiments demonstrate that the CD200-CD20R pathway enables two complementary and tissue-dependent strategies to limit atherogenesis: CD200 expression by bone-marrow derived cells limits systemic monocytosis, while CD200 expression by non-haematopoietic cells, e.g. endothelial cells, prevents local plaque growth. We show that CD200R signalling controls monopoiesis and macrophage activation through inhibiting phosphorylation of STAT1. Finally, CD200R expression on classical monocytes in peripheral blood of patients with coronary artery disease (CAD) is associated with a lower burden of CAD and a more favourable Virtual Histology plaque profile. Conclusions: The CD200 checkpoint is a key limiting factor for monopoiesis, monocyte-macrophage activation and recruitment in atherosclerosis with conserved features in human and mouse. It thus offers a novel potential therapeutic pathway to treat CVD.

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Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)–dependent migration and homing in multiple myeloma

Blood ◽

10.1182/blood-2006-07-035857 ◽

2006 ◽

Vol 109 (7) ◽

pp. 2708-2717 ◽

Cited By ~ 316

Author(s):

Yazan Alsayed ◽

Hai Ngo ◽

Judith Runnels ◽

Xavier Leleu ◽

Ujjal K. Singha ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Confocal Microscopy ◽

Bioluminescence Imaging ◽

Whole Body ◽

In Vivo Confocal Microscopy ◽

In Vivo Flow Cytometry ◽

Bone Marrow Niches

Abstract The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1–dependent migration was regulated by the PI3K and ERK/MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.

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The catabolism of intravenously injected heparan N-[35S]sulphate in the rat

Biochemical Journal ◽

10.1042/bj1660373 ◽

1977 ◽

Vol 166 (3) ◽

pp. 373-379 ◽

Cited By ~ 2

Author(s):

Martin A. Perry ◽

Gillian M. Powell ◽

Frederick S. Wusteman ◽

C. Gerald Curtis

Keyword(s):

Heparan Sulphate ◽

The Body ◽

Whole Body ◽

General Distribution ◽

In Vitro Experiments ◽

Metabolic Fate ◽

Inorganic Sulphate ◽

Anaesthetized Rats

The metabolic fate of heparan N-[35S]sulphate was studied in rats. Heparan sulphate was obtained from either bovine aorta or lung and labelled with 35S by desulphation and subsequent resulphation in vitro. Experiments in which heparan N-[35S]sulphate was administered intravenously to either free-range or wholly anaesthetized rats with ureter cannulae established that substantial desulphation occurs in vivo, with elimination of inorganic [35S]sulphate in urine. Oligosaccharides labelled with 35S, possible intermediates in heparan sulphate degradation, could not be detected in urine or blood. The general distribution of radioactivity after administration of heparan N-[35S]sulphate, as demonstrated by whole-body radioautography, suggested that desulphation was not restricted to one organ in particular. Support for this view was obtained in experiments in which heparan N-[35S]sulphate was administered to animals after the removal of kidneys, liver, spleen, pancreas or gastrointestinal tract. In all cases inorganic [35S]sulphate was still produced. The ability of rats of desulphate heparan N-[35S]sulphate was progressively impaired by increasing concentrations of heparin administered simultaneously. It was concluded that heparan sulphate is metabolized at a number of sites in the body by a sequence of degradative events leading to the formation of inorganic sulphate. It is also concluded that at least some of these events are common to heparan sulphate and heparin.

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Stoichiometry of two plant glycine decarboxylase complexes and comparison with a cyanobacterial glycine cleavage system

10.1101/2020.03.16.993188 ◽

2020 ◽

Author(s):

Maria Wittmiß ◽

Stefan Mikkat ◽

Martin Hagemann ◽

Hermann Bauwe

Keyword(s):

Mass Spectrometry ◽

Complex Formation ◽

Plant Mitochondria ◽

Glycine Decarboxylase ◽

Glycine Cleavage System ◽

Plant Leaf ◽

Molar Ratios ◽

Glycine Cleavage

ABSTRACTThe multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one-carbon compound 5,10-methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry-based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L2-4P2-8T-26H and 1L2-4P2-8T-20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1,550-1,650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS-P1 and GCS-L1 in comparison with GCS-P2 and GCS-L2, respectively, whereas the H-isoproteins GCS-H1 and GCS-H3 are fully redundant as indicated by their about equal amounts. Isoform GCS-H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins are present at low concentration but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H-protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes.

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