Drug information sources in professional work-a questionnaire study on physicians' usage and preferences (the drug information study).

PURPOSE: This study aimed to explore physicians' use of drug information in professional work, with special focus on those working in primary care, and also in relation to personal characteristics of physicians. METHODS: A web-based questionnaire was distributed by e-mail to physicians in five regions in Sweden. The questions concerned drug-related queries at issue when searching for information, sources used, and factors of importance for the choice of source, as well as responder characteristics. RESULTS: A total of 3254 (85%) out of 3814 responding physicians stated that they searched for drug information every week. For physicians working in primary health care, the corresponding number was 585 (96%). The most common drug-related issues searched for by 76% of physicians every week concerned pharmacotherapeutic aspects (e.g., dosing), followed by adverse drug reactions (63%). For 3349 (88%) physicians, credibility was the most important factor for the choice of sources of drug information, followed by easy access online (n = 3127, 82%). Further analyses among physicians in primary care showed that some personal characteristics, like seniority, sex, and country of education, as well as research experience, were associated with usage and preferences of drug information sources. CONCLUSIONS: This study confirms that physicians often use drug information sources in professional work, in particular those who work in primary health care. Credibility and easy access are key factors for usage. Among physicians in primary care, personal factors influenced the choice of drug information sources.

Heparanase - Discovery and Targets.

Heparanase was discovered during a study of the heparin proteoglycan (serglycin) in mast cells. Newly synthesized polysaccharide chains, kDa 60-100 x 103, were rapidly degraded to fragments similar in size to commercially available heparin (averaging 15 x 103). Analysis of the degradation products identified reducing-terminal glucuronic acid residues, shown by studies of heparin biosynthesis to be of ßD-configuration in the intact polymer. Heparanase, thus identified as an endo-ßD-glucuronidase, was subsequently identified in a variety of tissues and cells. The enzyme was subsequently implicated with a variety of pathophysiological processes, including in particular cancer, inflammatory diseases, and amyloidosis, as detailed in subsequent chapters of this volume. The target for enzyme action in these settings is primarily extracellular heparan sulfate proteoglycans; furthermore, intracellular cleavage initiates degradation of heparan sulfate chains by exolytic hydrolases and sulfatases, as part of normal turnover of the polysaccharide. More unexpectedly, heparanase also influences heparan sulfate biosynthesis, such that overexpression of the enzyme results in generation of highly sulfated, heparin-like oligosaccharides. The mechanism behind this effect remains unclear - along with the overall design of the molecular machinery in control of proteoglycan biosynthesis.

A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis.

Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.

Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response.

Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1ß (IL1ß) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1ß. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.

Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling.

Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

Drosophila heparan sulfate, a novel design.

Heparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.

Interacting polymer-modification enzymes in heparan sulfate biosynthesis.

Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcAß1-4GlcNSO3α1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluorescence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a functional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O-sulfated glucosamine residues. The inability to achieve simultaneous 2-O- and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme interactions in heparan sulfate biosynthesis.

Heparanase overexpression impairs inflammatory response and macrophage-mediated clearance of amyloid-ß in murine brain.

Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer's disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-ß peptide, implicated in Alzheimer's disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-ß elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer's disease.

Heparan sulfate domain organization and sulfation modulate FGF-induced cell signaling.

Heparan sulfates (HSs) modulate various developmental and homeostatic processes by binding to protein ligands. We have evaluated the structural characteristics of porcine HS in cellular signaling induced by basic fibroblast growth factor (FGF2), using CHO745 cells devoid of endogenous glycosaminoglycans as target. Markedly enhanced stimulation of cell signaling, measured as phosphorylation of ERK1/2 and protein kinase B, was only observed with the shortest HS chains isolated from liver, whereas the longer chains from either liver or intestine essentially prolonged duration of signals induced by FGF2 in the absence of polysaccharide. Structural analysis showed that contiguous sulfated domains were most abundant in the shortest HS chains and were more heavily sulfated in HS from liver than in HS from intestine. Moreover, the shortest chains from either source entered into ternary complexes with FGF2 and FGF receptor-1c more efficiently than the corresponding longer chains. In addition to authentic HSs, decasaccharide libraries generated by chemo-enzymatic modification of heparin were probed for effect on FGF2 signaling. Only the most highly sulfated decamers, previously found most efficient in ternary complex formation (Jastrebova, N., Vanwildemeersch, M., Rapraeger, A. C., Giménez-Gallego, G., Lindahl, U., and Spillmann, D. (2006) J. Biol. Chem. 281, 26884-26892), promoted FGF2 cellular signaling as efficiently as short HS chains from liver. Together these results suggest that the effects of HS on FGF2 signaling are determined by both the structure of the highly sulfated domains and by the organization/availability of such domains within the HS chain. These findings underpin the need for regulation of HS biosynthesis in relation to control of growth factor-induced signaling pathways.

Interactions between heparan sulfate and proteins: the concept of specificity.

Proteoglycan (PG) coreceptors carry heparan sulfate (HS) chains that mediate interactions with growth factors, morphogens, and receptors. Thus, PGs modulate fundamental processes such as cell survival, division, adhesion, migration, and differentiation. This review summarizes recent biochemical and genetic information that sheds new light on the nature of HS-protein binding. Unexpectedly, many interactions appear to depend more on the overall organization of HS domains than on their fine structure.

Heparanase overexpression impedes perivascular clearance of amyloid-ß from murine brain: relevance to Alzheimer's disease.

Defective amyloid-ß (Aß) clearance from the brain is a major contributing factor to the pathophysiology of Alzheimer's disease (AD). Aß clearance is mediated by macrophages, enzymatic degradation, perivascular drainage along the vascular basement membrane (VBM) and transcytosis across the blood-brain barrier (BBB). AD pathology is typically associated with cerebral amyloid angiopathy due to perivascular accumulation of Aß. Heparan sulfate (HS) is an important component of the VBM, thought to fulfill multiple roles in AD pathology. We previously showed that macrophage-mediated clearance of intracortically injected Aß was impaired in the brains of transgenic mice overexpressing heparanase (Hpa-tg). This study revealed that perivascular drainage was impeded in the Hpa-tg brain, evidenced by perivascular accumulation of the injected Aß in the thalamus of Hpa-tg mice. Furthermore, endogenous Aß accumulated at the perivasculature of Hpa-tg thalamus, but not in control thalamus. This perivascular clearance defect was confirmed following intracortical injection of dextran that was largely retained in the perivasculature of Hpa-tg brains, compared to control brains. Hpa-tg brains presented with thicker VBMs and swollen perivascular astrocyte endfeet, as well as elevated expression of the BBB-associated water-pump protein aquaporin 4 (AQP4). Elevated levels of both heparanase and AQP4 were also detected in human AD brain. These findings indicate that elevated heparanase levels alter the organization and composition of the BBB, likely through increased fragmentation of BBB-associated HS, resulting in defective perivascular drainage. This defect contributes to perivascular accumulation of Aß in the Hpa-tg brain, highlighting a potential role for heparanase in the pathogenesis of AD.

Heparan sulfate mediates amyloid-beta internalization and cytotoxicity.

Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.

Transgenic or tumor-induced expression of heparanase upregulates sulfation of heparan sulfate.

Heparan sulfate proteoglycans (HSPGs) interact with numerous proteins of importance in animal development and homeostasis. Heparanase, which is expressed in normal tissues and upregulated in angiogenesis, cancer and inflammation, selectively cleaves beta-glucuronidic linkages in HS chains. In a previous study, we transgenically overexpressed heparanase in mice to assess the overall effects of heparanase on HS metabolism. Metabolic labeling confirmed extensive fragmentation of HS in vivo. In the current study we found that in liver showing excessive heparanase overexpression, HSPG turnover is accelerated along with upregulation of HS N- and O-sulfation, thus yielding heparin-like chains without the domain structure typical of HS. Heparanase overexpression in other mouse organs and in human tumors correlated with increased 6-O-sulfation of HS, whereas the domain structure was conserved. The heavily sulfated HS fragments strongly promoted formation of ternary complexes with fibroblast growth factor 1 (FGF1) or FGF2 and FGF receptor 1. Heparanase thus contributes to regulation of HS biosynthesis in a way that may promote growth factor action in tumor angiogenesis and metastasis.

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling.

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

Low molecular weight heparin stimulates myometrial contractility and cervical remodeling in vitro.

OBJECTIVES: The low molecular weight heparin, Dalteparin, shortens human labor time. The aim of this study was to investigate if the mechanism behind this effect involves myometrial contractility and cervical ripening and if the anticoagulative activity is necessary for its effect. DESIGN: Experimental in vitro study. SETTING: Lund University and Karolinska Institute, Sweden. METHODS: The effect of low molecular weight heparins with or without anticoagulative properties on myometrial contractility was measured in vitro on smooth muscle strips from biopsies obtained at elective cesarean sections. The effects on cervical ripening were assessed in cervical fibroblasts cultured from explants of cervical biopsies obtained at delivery. MAIN OUTCOME MEASURES: Mean force and number of contractions in uterine smooth muscle strips and interleukin-8 (IL-8) secretion in cervical fibroblasts. RESULTS: Myometrial smooth muscle strips pretreated with low molecular weight heparins showed increased contractile activity compared to untreated smooth muscle strips. Secretion of IL-8 from cultured cervical fibroblasts was significantly increased after treatment with low molecular weight heparin. Both these effects were independent of anticoagulative activity of the low molecular weight heparin. CONCLUSIONS: A possible underlying mechanism for the shortened labor time after low molecular weight heparin treatment is enhanced myometrial contractility and an increased IL-8 secretion in cervical fibroblast, mimicking the final cervical ripening in vivo. Our data support the notion that anticoagulant activity is not required to promote labor.

Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells.

Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.

Specificity of glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) interact with a variety of proteins with important functions in development and homeostasis. Most of these proteins bind to heparin in vitro, a highly sulfated GAG species, although heparan sulfate and/or chondroitin/dermatan sulfate are more frequent physiological ligands. Binding affinity and specificity are determined by charge distribution, mainly due to sulfate and carboxylate groups and by GAG chain conformation. Interactions may be nonspecific, essentially reflecting charge density or highly specific, dependent on rare GAG-structural features. Yet other GAG epitopes bind protein ligands with intermediate specificity and variable affinity. Studies of heparan sulfate biosynthesis point to stochastic but strictly regulated, cell-specific polymer modification. Together, these features allow for graded modulation of protein functional response.

Heparan sulfate-protein interactions--a concept for drug design?

The glycosaminoglycan, heparan sulfate (HS) is composed of alternating units of hexuronic acid and glucosamine, that are variously sulfate-substituted at different positions. Proteoglycans carrying HS chains are ubiquitously expressed at cell surfaces and in the extracellular matrix. The structures of these chains are highly variable, yet under strict biosynthetic control. Due to their high negative charge, HS chains interact with a multitude of proteins, including growth factors/morphogens and their receptors, chemokines, and extracellular-matrix proteins. These interactions regulate key events in embryonic development and in homeostasis. HS-protein interactions vary with regard to specificity, and often seem to depend primarily on charge density rather than on strict carbohydrate sequence. The organization of sulfated domains along the HS chain appears to be of importance. HS-protein interactions are involved in a variety of pathophysiological processes, including inflammation, angiogenesis, and amyloid deposition. Drugs targeting such interactions may be useful in treatment of disease conditions as diverse as cancer, inflammatory bowel disease, and Alzheimer's disease. Potential drugs may mimick HS oligosaccharides, but could also be peptides blocking the protein-binding domains of HS chains. Drug generation requires a firm understanding of the pathophysiological role of a given HS-protein interaction, and of the aspect of specificity. Even inhibition of HS biosynthesis may be considered.

Fibroblast growth factors share binding sites in heparan sulphate.

HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs.

Enzyme overexpression - an exercise toward understanding regulation of heparan sulfate biosynthesis.

Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.