M R el-Maghrabi

Hackensack University Medical Center, Hackensack, New Jersey, United States

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Publications (105)445.48 Total impact

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    ABSTRACT: The authors have recently demonstrated that increased gene expression of glucose-6-phosphatase (Glu-6-Pase) in hemorrhagic hypotension (HH) and following lactated Ringer's resuscitation (LR) is associated with a decrease in insulin and an increase in corticosterone concentrations. Objective: To evaluate the in-vivo role of hormones the authors used insulin (IN), phentolamine and propranolol (PP) as an adrenergic blocker, and cyclic somatostatin (CS) as a glucagon blocker to prevent the induction of Glu-6-Pase gene expression in liver and kidney following HH and LR. Methods: Hemorrhage was induced in fasted anesthetized rats, and the reduction of blood pressure to 40 mm Hg for a duration of 30 minutes was accomplished by withdrawal or infusion of shed blood. The resuscitated group underwent hemorrhage followed by fluid resuscitation with lactated Ringer's solution. Results: Neither PP nor CS treatment could block the induction of Glu-6-Pase messenger ribonucleic acid (mRNA) following either HH or LR. However, the administration of IN significantly prevented the increase of Glu-6-Pase mRNA level and activity in both liver and kidney following HH and LR. This was associated with a normalization of plasma glucose, corticosterone, and glucagon levels and glucose-6-phosphate concentrations in liver and kidney toward prehemorrhage levels. Conclusions: These results indicate that in-vivo treatment with insulin during hemorrhagic hypotension and resuscitation is capable of preventing the increase in Glu-6-Pase gene expression in liver and kidney responsible for the observed hyperglycemia.
    Academic Emergency Medicine 01/2008; 7(7):731 - 738. DOI:10.1111/j.1553-2712.2000.tb02259.x · 2.20 Impact Factor
  • Song-Gun Kim, Michael Cavalier, M Raafat El-Maghrabi, Yong-Hwan Lee
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    ABSTRACT: To understand the molecular basis of a phosphoryl transfer reaction catalyzed by the 6-phosphofructo-2-kinase domain of the hypoxia-inducible bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), the crystal structures of PFKFB3AMPPCPfructose-6-phosphate and PFKFB3ADPphosphoenolpyruvate complexes were determined to 2.7 A and 2.25 A resolution, respectively. Kinetic studies on the wild-type and site-directed mutant proteins were carried out to confirm the structural observations. The experimentally varied liganding states in the active pocket cause no significant conformational changes. In the pseudo-substrate complex, a strong direct interaction between AMPPCP and fructose-6-phosphate (Fru-6-P) is found. By virtue of this direct substrate-substrate interaction, Fru-6-P is aligned with AMPPCP in an orientation and proximity most suitable for a direct transfer of the gamma-phosphate moiety to 2-OH of Fru-6-P. The three key atoms involved in the phosphoryl transfer, the beta,gamma-phosphate bridge oxygen atom, the gamma-phosphorus atom, and the 2-OH group are positioned in a single line, suggesting a direct phosphoryl transfer without formation of a phosphoenzyme intermediate. In addition, the distance between 2-OH and gamma-phosphorus allows the gamma-phosphate oxygen atoms to serve as a general base catalyst to induce an "associative" phosphoryl transfer mechanism. The site-directed mutant study and inhibition kinetics suggest that this reaction will be catalyzed most efficiently by the protein when the substrates bind to the active pocket in an ordered manner in which ATP binds first.
    Journal of Molecular Biology 07/2007; 370(1):14-26. DOI:10.1016/j.jmb.2007.03.038 · 3.96 Impact Factor
  • Song-Gun Kim, Nathan P Manes, M Raafat El-Maghrabi, Yong-Hwan Lee
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    ABSTRACT: The hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) plays a crucial role in the progression of cancerous cells by enabling their glycolytic pathways even under severe hypoxic conditions. To understand its structural architecture and to provide a molecular scaffold for the design of new cancer therapeutics, the crystal structure of the human form was determined. The structure at 2.1 A resolution shows that the overall folding and functional dimerization are very similar to those of the liver (PFKFB1) and testis (PFKFB4) forms, as expected from sequence homology. However, in this structure, the N-terminal regulatory domain is revealed for the first time among the PFKFB isoforms. With a beta-hairpin structure, the N terminus interacts with the 2-Pase domain to secure binding of fructose-6-phosphate to the active pocket, slowing down the release of fructose-6-phosphate from the phosphoenzyme intermediate product complex. The C-terminal regulatory domain is mostly disordered, leaving the active pocket of the fructose-2,6-bisphosphatase domain wide open. The active pocket of the 6-phosphofructo-2-kinase domain has a more rigid conformation, allowing independent bindings of substrates, fructose-6-phosphate and ATP, with higher affinities than other isoforms. Intriguingly, the structure shows an EDTA molecule bound to the fructose-6-phosphate site of the 6-phosphofructo-2-kinase active pocket despite its unfavorable liganding concentration, suggesting a high affinity. EDTA is not removable from the site with fructose-6-P alone but is with both ATP and fructose-6-P or with fructose-2,6-bisphosphate. This finding suggests that a molecule in which EDTA is covalently linked to ADP is a good starting molecule for the development of new cancer-therapeutic molecules.
    Journal of Biological Chemistry 03/2006; 281(5):2939-44. DOI:10.1074/jbc.M511019200 · 4.60 Impact Factor
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    Subir R Maitra, Sikha Bhaduri, M Raafat El-Maghrabi, Marc J Shapiro
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    ABSTRACT: Hemorrhage initiates an inflammatory response that induces the systemic release of cytokines and sequestration of polymorphonuclear neutrophils. Sequestered polymorphonuclear neutrophils release proteases, including matrix metalloproteinases (MMPs) that degrade elements of the extracellular matrix, contributing to the morbidity and mortality seen from hemorrhage. Activation of MMPs may be associated with changes in transforming growth factor beta1 (TGF-beta1) and caspase-3 signaling pathways. In this study, the authors examined hemorrhage-induced changes in the expression of rat hepatic MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-l), TGF-beta1, and caspase-3 activities in the presence and absence of the MMP inhibitor hydroxamate. Hemorrhagic shock was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure level of 40 mm Hg, maintained for 90 minutes by withdrawal and infusion of blood, followed by a resuscitation period of lactated Ringer's infusion. Rats received either hydroxamate (25 mg/kg) or vehicle by gavage before hemorrhage. Twenty-four hours after resuscitation, plasma and liver samples were collected. Liver MMP-9, TGF-beta1, and caspase-3 levels were quantified by Western immunoblotting. Plasma glutamic oxaloacetic transaminase (GOT) and plasma glutamic pyruvic transaminase (GPT) were determined enzymatically. Plasma GOT, plasma GPT, and liver MMP-9, TGF-beta1, and caspase-3 levels were all significantly elevated at 24 hours postresuscitation when compared with the control values. Hepatic TIMP-1, an in vivo inhibitor of MMP-9, was unaltered at 24 hours. Hydroxamate treatment reduced GOT, GPT, MMP-9, TGF-beta1, and caspase-3 levels at 24 hours. The mortality of hemorrhaged untreated rats was 29% after 24 hours, and pretreatment with hydroxamate reduced mortality to 0%. These results indicate the beneficial effects of MMP inhibitor in preventing an increase in GOT, GPT, MMP-9, TGF-beta1, and caspase-3 activity with the potential for improvement of hepatic injury due to hemorrhage.
    Academic Emergency Medicine 10/2005; 12(9):797-803. DOI:10.1197/j.aem.2005.04.017 · 2.20 Impact Factor
  • Subir R Maitra, Marc J Shapiro, Sikha Bhaduri, M Raafat El-Maghrabi
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    ABSTRACT: We have previously demonstrated that hepatic matrixmetalloproteinase (MMP)-9 and gelatinase activity increased significantly after sepsis, and pretreatment with chemically modified tetracycline (CMT-3) inhibited these expressions and improved survivability. It has been established that MMP-9 release from hepatic nonparenchymal cells activates transforming growth factor (TGF)-beta1, which in turn catalyzes the conversion of procaspase-8 into active caspase-8. Caspase-8 activates caspase-3, which in turn degrades fibronectin and focal adhesion kinase and leads to disruption of hepatic architecture and integrity. We have been interested in investigating the role of posttreatment with CMT-3 on hepatic MMP-9, TGF-beta1, and caspase-3 activity following sepsis. Laboratory experiment. University laboratory. Male Sprague-Dawley rats. In this study, sepsis was induced in rats by cecal ligation and puncture (CLP), and 2 hrs later, half of the rats received CMT-3 (25 mg/kg), whereas the other half received vehicle by gavage. Twenty-four and 48 hrs after sepsis induction, blood and liver samples were collected. Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined by enzymatic method, and the activation states of hepatic MMP-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-beta1, and caspase-3 were determined by Western immunoblotting. Plasma GOT, GPT, and hepatic MMP-9 activity increased 2.5-fold, and TFG-beta1 and caspase-3 activity increased 1.5- to 2-fold at 24 hrs and 48 hrs post-CLP; CMT-3 treatment blocked these increases. Furthermore, CMT-3 treatment also led to increased TIMP-1 level, an in vivo inhibitor of MMP-9. MMP-2 level was unaffected by CLP. The 24-hr and 48-hr mortality rates for CLP rats were 29% and 50%, whereas posttreatment with CMT-3 resulted in 0% mortality. Our results are consistent with an MMP-9-induced caspase-3 activation in response to CLP. CMT-3 posttreatment increased TIMP-1 level and thereby inhibited MMP-9, which in turn decreased TGF-beta1 and caspase-3 signaling pathways and improved survivability in septic rats.
    Critical Care Medicine 08/2005; 33(7):1577-81. · 6.15 Impact Factor
  • Nathan P Manes, M Raafat El-Maghrabi
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    ABSTRACT: The two enzymatic activities of the highly conserved catalytic core of 6PF2K/Fru-2,6-P(2)ase are thought to be reciprocally regulated by the amino- and carboxy-terminal regions unique to each isoform. In this study, we describe the recombinant expression, purification, and kinetic characterization of two human brain 6PF2K/Fru-2,6-P(2)ase splice variants, HBP1 and HBP2. Interestingly, both lack an arginine which is highly conserved among other tissue isoforms, and which is understood to be critical to the fructose-2,6-bisphosphatase mechanism. As a result, the phosphatase activity of both HBP isoforms is negligible, but we found that it could be recovered by restoration of the arginine by site directed mutagenesis. We also found that AMP activated protein kinase and protein kinases A, B, and C catalyzed the phosphorylation of Ser-460 of HBP1, and that in addition both isoforms are phosphorylated at a second, as yet undetermined site by protein kinase C. However, none of the phosphorylations had any effect on the intrinsic kinetic characteristics of either enzymatic activity, and neither did point mutation (mimicking phosphorylation), deletion, and alternative-splice modification of the HBP1 carboxy-terminal region. Instead, these phosphorylations and mutations decreased the sensitivity of the 6PF2K to a potent allosteric inhibitor, phosphoenolpyruvate, which appears to be the major regulatory mechanism.
    Archives of Biochemistry and Biophysics 07/2005; 438(2):125-36. DOI:10.1016/j.abb.2005.04.011 · 3.04 Impact Factor
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    ABSTRACT: Objectives: We have previously demonstrated that hepatic matrixmetalloproteinase (MMP)-9 and gelatinase activity increased significantly after sepsis, and pretreatment with chemically modified tetracycline (CMT-3) inhibited these expressions and improved survivability. It has been established that MMP-9 release from hepatic nonparenchymal cells activates transforming growth factor (TGF)-beta 1, which in turn catalyzes the conversion of procaspase-8 into active caspase-8. Caspase-8 activates caspase-3, which in turn degrades fibronectin and focal adhesion kinase and leads to disruption of hepatic architecture and integrity. We have been interested in investigating the role of post-treatment with CMT-3 on hepatic MMP-9, TGF-beta 1, and caspase-3 activity following sepsis. Design: Laboratory experiment. Setting: University laboratory. Subjects: Male Sprague-Dawley rats. Interventions: In this study, sepsis was induced in rats by cecal ligation and puncture (CLP), and 2 hrs later, half of the rats received CMT-3 (25 mg/kg), whereas the other half received vehicle by gavage. Twenty-four and 48 hrs after sepsis induction, blood and liver samples were collected. Measurements and Main Results. Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined by enzymatic method, and the activation states of hepatic MMP-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-beta 1, and caspase-3 were determined by Western immunoblotting. Plasma GOT, GPT, and hepatic MMP-9 activity increased 2.5-fold, and TFG-beta 1 and caspase-3 activity increased 1.5- to 2-fold at 24 hrs and 48 hrs post-CLP; CMT-3 treatment blocked these increases. Furthermore, CMT-3 treatment also led to increased TIMP-1 level, an in vivo inhibitor of MMP-9. MMP-2 level was unaffected by CLP, The 24-hr and 48-hr mortality rates for CLP rats were 29% and 50%, whereas post-treatment with CMT-3 resulted in 0% mortality. Conclusions. Our results are consistent with an MMP-9-induced caspase-3 activation in response to CLP. CMT-3 post-treatment increased TIMP-1 level and thereby inhibited MMP-9, which in turn decreased TGF-beta 1 and caspase-3 signaling pathways and improved survivability in septic rats.
    Critical Care Medicine 07/2005; 33(7):1577-1581. DOI:10.1097/01.CCM.0000169880.82060.F7 · 6.15 Impact Factor
  • S R Maitra, S Bhaduri, M R El-Maghrabi, M J Shapiro
    Shock 01/2004; 21:46. DOI:10.1097/00024382-200406002-00136 · 2.73 Impact Factor
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    ABSTRACT: The phosphorylation states and the inferred activation of specific members of the mitogen-activated protein kinase (MAPK) pathways (p42/44, p38, and SAPK/JNK) were quantitated in the livers of rats by Western blot analysis during the progression of sepsis. In addition, the authors examined the effects of insulin and mifepristone (RU-486) administration on these signal transduction pathways during sepsis. Sepsis was brought about by the cecal ligation and puncture (CLP) method. The control group underwent sham operation. One experimental group was injected with insulin (0.5 U/kg, IV); prior to the CLP or sham procedure, the second group was injected with RU-486, a glucocorticoid antagonist (mifepristone, 20 mg/kg, IP), and the third group was injected with saline (control). The activities of SAPK/JNK were unaltered throughout the course of sepsis, while those of p42/44 MAPK increased four-fold after 24 hours of sepsis. The activation of p38 MAPK was also increased after 0.5 and 1.5 hours of sepsis, but returned to normal during late sepsis (24 hours). Rats that had received RU-486 before CLP showed no change in p42/44 MAPK compared with the untreated group, but the drug appeared to suppress the activation after 24 hours post-CLP. SAPK/JNK activation, on the other hand, was slightly enhanced by RU-486 pretreatment after 1.5 and 24 hours of CLP compared with the untreated CLP and the treated sham-operated rats. Insulin, given preoperatively, had no effect on the activation of SAPK/JNK, although p42/44 MAPK pathways were activated by the hormone at 0.5 hours and 1.5 hours post-CLP only, but not in the sham-operated controls or at 24 hours post-CLP. The p38 MAPK activation seen after 0.5 and 1.5 hours of CLP was suppressed by RU-486. Insulin pretreatment appears to suppress the phosphorylation of p38 in both the CLP and sham-operated groups. The data suggest that sepsis induces two phases of MAPK activation, an early phase in which p38 is active, followed by a second, more pronounced phase of p42/44 MAPK activation that occurs only late in sepsis. Hormonal administration results suggest that RU-486 suppresses the activation of p38 seen in early sepsis, and the activation of p42/44 MAPK seen in late sepsis.
    Academic Emergency Medicine 02/2003; 10(1):1-8. DOI:10.1197/aemj.10.1.1 · 2.20 Impact Factor
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    ABSTRACT: Recent studies have suggested that cell-to-cell coupling, which occurs via gap junctions, may play a role in CO(2) chemoreception. Here, we used immunoblot and immunohistochemical analyses to investigate the presence, distribution, and cellular localization of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) in putative CO(2)-chemosensitive brainstem regions in both neonatal and adult rats. Immunoblot analyses revealed that both Cx subtypes were expressed in putative CO(2)-chemosensitive brainstem regions; however, regional differences in expression were observed. Immunohistochemical experiments confirmed Cx expression in each of the putative CO(2)-chemosensitive brainstem regions, and further demonstrated that Cx26 and Cx32 were found in neurons and Cx26 was also found in astrocytes in these regions. Thus, our findings suggest the potential for gap junctional communication in these regions in both neonatal and adult rats. We propose that the gap junction proteins Cx26 and Cx32, at least in part, form the neuroanatomical substrate for this gap junctional communication, which is hypothesized to play a role in central CO(2) chemoreception.
    Respiration Physiology 01/2002; 129(1-2):101-21. DOI:10.1016/S0034-5687(01)00299-7
  • M. Raafat El-Maghrabi, Frank Noto, Ning Wu, Nathan Manes
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    ABSTRACT: The present review addresses recent advances in research into a family of bifunctional enzymes that are responsible for the twofold task of synthesizing and hydrolyzing fructose-2,6-bisphosphate (Fru-2,6-P2), which in turn regulates the rate of glycolysis in most cells. The structure of the synthetic kinase, conjoined at its carboxyl-terminus to the phosphatase, is very highly conserved throughout evolution and differentiation, with isotypic expression arising from highly variable amino-terminal and carboxyl-terminal regulatory domains. These domains, which frequently contain protein-kinase-catalyzed phosphorylation motifs, are responsible for the widely divergent kinetics observed in various tissues and species, and for the hormonal modulation that alters intracellular levels of Fru-2,6-P2. The present review discusses recent advances in relating structure to function, and the identification of new pathways of transcriptional regulation of this important family of regulatory enzymes.
    Current Opinion in Clinical Nutrition and Metabolic Care 10/2001; 4(5):411-8. DOI:10.1097/00075197-200109000-00012 · 3.97 Impact Factor
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    ABSTRACT: The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.
    Circulation Research 07/2001; 88(12):E84-7. · 11.09 Impact Factor
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    ABSTRACT: In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a b subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a b subunit for the cardiac delayed rectifier IKr, these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001;88:e84-e87.)
    Circulation Research 06/2001; 88(12). DOI:10.1161/hh1201.093511 · 11.09 Impact Factor
  • S R Maitra, S Wang, M R El-Maghrabi
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    ABSTRACT: To assess the role of glucocorticoid receptor antagonists and mediators released by Kupffer cells and other resident macrophages, we have used RU486 and gadolinium chloride to prevent the induction of glucose-6-phosphatase (Glu-6-Pase) gene expression in the liver following hemorrhagic shock (HS) and lactated Ringer's (LR) solution resuscitation. HS was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure of 40 mmHg and maintained for 30 min by withdrawal or infusion of blood. The LR solution group underwent induction and maintenance of HS for 30 min followed by LR resuscitation. Rats were injected with gadolinium chloride (7 mg/kg) to inhibit the phagocytic function of Kupffer cells, and with glucocorticoid receptor antagonist RU486 (20 mg/kg) prior to induction of HS. Arterial blood samples were obtained and livers were freeze clamped in liquid nitrogen and stored at -70 degrees C for subsequent analysis. Northern blot analysis indicated that Glu-6-Pase mRNA abundance increased 2-fold in HS rats and a further 2-fold with resuscitation. Gadolinium chloride administration had no significant effect on Glu-6-Pase mRNA abundance in HS or in LR solution. In contrast, RU486 pre-treatment reduced Glu-6-Pase mRNA by about one half in HS rats compared with control and that in LR solution to normal. This was associated with a normalization of Glu-6-Pase activity and plasma glucose toward pre-hemorrhage levels. These results suggest that gadolinium chloride inhibition of macrophage factor release has no effect on the induction of Glu-6-Pase mRNA during HS or in LR solution resuscitation. On the other hand, the suppression of Glu-6-Pase mRNA by RU486 suggests that glucocorticoids are responsible for the induction of the mRNA in HS and during LR resuscitation. KEYWORDS-Shock, hyperglycemia, corticosterone, gadolinium chloride, diltiazem, animal model, mRNA
    Shock 12/2000; 14(5):578-81. · 2.73 Impact Factor
  • S R Maitra, SY Wang, C E Brathwaite, M R El-Maghrabi
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    ABSTRACT: The influence of sepsis on the expression and activity of hepatic glucose-6-phosphatase (Glu-6-Pase) was examined during the early hyperglycemic phase and the later hypoglycemic phase. Sepsis was induced in anesthetized, fasted rats by cecal ligation and puncture, and liver samples were taken at 0, 0.5, 1, 1.5, and 20 hours after cecal ligation and puncture. The mRNA abundance of hepatic Glu-6-Pase increased fourfold at 0.5 hours over healthy control values, two-fold after 1 hour, and returned to normal after 1.5 hours. This finding was followed by a corresponding increase in Glu-6-Pase activity and was coincident with increased plasma glucose levels and decreased liver glucose-6-phosphate (Glu-6-P) at 0.5 and 1 hours. Plasma insulin and glucagon levels remained unchanged during this period, whereas corticosterone levels increased 2.5-fold over control values. At 20 hours cecal ligation and puncture, plasma glucose levels returned to normal, coincident with a 90% reduction in Glu-6-Pase mRNA abundance. Glu-6-Pase activity and Glu-6-P concentration returned to normal levels, while insulin, glucagon, and corticosterone levels increased significantly, i.e., 40-fold, 6.5-fold, and 6-fold, respectively. The initial rise and subsequent decline in blood glucose correlate very well with a corticosterone-dependent induction of hepatic Glu-6-Pase, mRNA, and protein, followed by an insulin-dependent suppression of its expression.
    The Journal of trauma 08/2000; 49(1):38-42. DOI:10.1097/00005373-200007000-00005 · 2.96 Impact Factor
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    Irwin J. Kurland, Brett Chapman, M. Raafat El-Maghrabi
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    ABSTRACT: Liver and skeletal muscle isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P(2)ase) isoenzymes are products of alternatively spliced first exons of the same gene, with common kinase and bisphosphatase domains. The muscle-specific exon-1 encodes nine unique amino acids, that lack the cAMP-dependent protein kinase (PK-A) phosphorylation site, and differ in sequence from those encoded by the liver-specific exon-1 (32 amino acids), contributing to its much lower affinity for fructose 6-phosphate (Fru-6-P). PK-A phosphorylation of the liver isoform at Ser(32) reduces the affinity of the kinase for Fru-6-P, and stimulates the bisphosphatase V(max). In the present study, we have defined the locus of interaction of the N-terminal residues with the N-terminal kinase and C-terminal domains by successive N- and C-terminal deletions. This study shows that: (1) residues Gly(5)-Glu(6)-Leu(7) of the liver isoform are responsible for increasing the affinity of 6PF2K for Fru-6-P, maintaining the inhibition of Fru-2,6-P(2)ase activity, and mediating the effects of PK-A phosphorylation on the two activities; (2) the loss of Fru-6-P inhibition of the bisphosphatase and the enhancement of its V(max), rather than the inhibition of the kinase, may be responsible for the behaviour of the muscle isoform primarily as a bisphosphatase; (3) the composition of residues 24-32 of the liver form appears to confer the enhanced kinase catalytic rate of this form over that of the muscle isoform. It is concluded that specific regions of the N-terminus of liver and skeletal muscle 6PF2K/Fru-2,6-P(2)ase have a role in adapting the two activities to work in the physiological range of pH and substrate concentrations found in each particular tissue.
    Biochemical Journal 05/2000; 347(Pt 2):459-67. DOI:10.1042/0264-6021:3470459 · 4.78 Impact Factor
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    Ying Zhang, M. Raafat El-Maghrabi, FA Gomez
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    ABSTRACT: The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions.
    The Analyst 05/2000; 125(4):685-8. DOI:10.1039/A909092D · 3.91 Impact Factor
  • T Wang, L Dowal, M R El-Maghrabi, M Rebecchi, S Scarlata
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    ABSTRACT: Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.
    Journal of Biological Chemistry 04/2000; 275(11):7466-9. · 4.60 Impact Factor
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    ABSTRACT: Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the βγ subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-β2 (PLC-β2), which is regulated by Gβγ, replaces the PH domain of PLC-δ1 which binds to, but is not regulated by, Gβγ. We found that attachment of the PH domain of PLC-β2 onto PLC-δ1 not only causes the membrane-binding properties of PLC-δ1 to become similar to those of PLC-β2, but also results in a Gβγ-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.
    Journal of Biological Chemistry 03/2000; 275(11):7466-7469. · 4.60 Impact Factor
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    ABSTRACT: Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.
    Biochemistry 03/2000; 39(5):1169-79. DOI:10.1021/bi992159y · 3.19 Impact Factor

Publication Stats

3k Citations
445.48 Total Impact Points

Institutions

  • 2008
    • Hackensack University Medical Center
      Hackensack, New Jersey, United States
  • 1994–2007
    • Stony Brook University
      • • Department of Physiology & Biophysics
      • • Department of Emergency Medicine
      Stony Brook, New York, United States
  • 1986–2005
    • State University of New York
      New York City, New York, United States
  • 2000–2003
    • Stony Brook University Hospital
      Stony Brook, New York, United States
    • University of California, Los Angeles
      • Molecular Biology Institute
      Los Ángeles, California, United States
  • 1989–1995
    • University of Barcelona
      Barcino, Catalonia, Spain
  • 1986–1995
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 1993
    • Public Health Agency
      Béal Feirste, N Ireland, United Kingdom
  • 1981–1989
    • Vanderbilt University
      • Department of Molecular Physiology and Biophysics
      Nashville, MI, United States
  • 1988
    • Wake Forest University
      • Department of Biochemistry
      Winston-Salem, North Carolina, United States