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R A Kahn

Publications and source records attributed to R A Kahn.

At least 55 records · Page 3Linked to original sources
PubMed1997

Modulation of ischemic excitatory neurotransmitter and gamma-aminobutyric acid release during global temporary cerebral ischemia by selective neuronal nitric oxide synthase inhibition.

Nitric oxide release during cerebral ischemia is the result of both neuronal and endothelial subclasses of nitric oxide synthase (NOS). In this study, we examined the role of specific neuronal NOS inhibition (nNOSI) on excitatory neurotransmitter and gamma-aminobutyric acid (GABA) release during global cerebral ischemia. Microdialysis probes were placed into the striatum of 24 rats. After probe stabilization, rats were randomized to receive 7-nitroindazole (7-NI), a selective nNOSI, in doses of 0, 5, 10, or 20 mg/kg. Temporary global forebrain ischemia was induced for 15 min, followed by 60 min of reperfusion. nNOSI administration did not produce detectable changes in neurotransmitter recovery prior to ischemia. There were significant increases in aspartate (ASP), glutamate (GLU), glycine (GLY), and GABA recovery during ischemia in the absence of nNOSI. 7-NI resulted in an attenuation in GLU, GLY, and GABA recovery during ischemia and reperfusion. No differences in ASP recovery were detected with nNOSI. Differences between the present study and other studies that examine the role of nonspecific constitutive NOSI during cerebral ischemia demonstrate the contribution of neuronal NOS on the modulation of ischemic excitatory neurotransmitter and GABA release.

Animals↗
PubMed1997

Modulation of ischemic excitatory neurotransmitter and gamma-aminobutyric acid release during global temporary cerebral ischemia by local nitric oxide synthase inhibition.

Systemic nitric oxide synthase inhibition (NOSI) decreases cerebral blood flow, which may worsen ischemic insults. To examine the local effects of NOSI without this confounding effect, we examined the role of a locally administered NOSI, NG-nitro-L-arginine-methyl-ester (L-NAME), on neurotransmitter recovery during cerebral ischemia. Rats were assigned to one of three groups: locally administered L-NAME via a striatal microdialysis probe (n = 11), systemic L-NAME (n = 5), or control (n = 11). Temporary global forebrain ischemia was induced for 15 min, followed by 60 min of reperfusion. L-NAME resulted in decreases of basal aspartate (ASP; 74% of basal) and glutamate (GLU; 60% of basal) recovery. While systemic L-NAME caused significant increases in ischemic ASP and GLU recovery (by 224% and 110%, respectively, compared with ischemic controls), local NOSI administration resulted in a significant attenuation of peak ASP, GLU, glycine, and gamma-aminobutyric acid recovery (43%, 38%, 53%, and 72%, respectively, compared with ischemic controls). We conclude that local NOSI attenuated ischemic neurotransmitter recovery during ischemia/reperfusion. Our results emphasize the importance of the systemic effects of NOSI and suggest both deleterious and beneficial effects of NOSI during ischemia/reperfusion.

Animals↗
PubMed1997

Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum.

A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled.

Amino Acid Sequence↗
PubMed1996

Isolation and reconstitution of the heme-thiolate protein obtusifoliol 14alpha-demethylase from Sorghum bicolor (L.) Moench.

The heme-thiolate (cytochrome P450) enzyme which catalyzes the 14alpha-demethylation of obtusifoliol has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The obtusifoliol 14alpha-demethylase is a key enzyme in plant sterol biosynthesis and a target for the design of phyla-specific sterol 14alpha-demethylase inhibitors. Microsomal cytochrome P450s were solubilized by using the detergents Renex 690 and reduced Triton X-100, and the obtusifoliol 14alpha-demethylase was isolated by DEAE ion exchange and dye affinity column chromatography. The isolated enzyme has an absorption spectrum characteristic for low spin cytochrome P450s and produces a Type I binding spectrum with obtusifoliol as substrate. Binding spectra were not obtained with lanosterol, campesterol, sitosterol, or stigmasterol. Obtusifoliol 14alpha-demethylase has an apparent molecular mass of 53 kDa and is estimated to constitute approximately 20% of the total cytochrome P450 content of the microsomal membranes and about 0.2% of the total microsomal protein. Gas chromatography-mass spectrometry analysis of reconstitution experiments with dilauroylphosphatidylcholine micelles containing isolated obtusifoliol 14alpha-demethylase and sorghum NADPHcytochrome P450 oxidoreductase demonstrated the conversion of obtusifoliol (4alpha,14alpha-dimethyl-5alpha-ergosta-8, 24(28)-dien-3beta-ol) to 4alpha-methyl-5alpha-ergosta-8,14, 24(28)-trien3beta-ol, the 14alpha-demethylated product of obtusifoliol with a double bond introduced at the Delta14 position. The N-terminal amino acid sequence of the protein is MDLADIPQ/KQQRLMAGXALVV. Five internal sequences were obtained after endoproteinase Lys-C and Glu-C digestion. The fragment AAGAFSYISFGGGRH aligns with the unique heme binding domain of mammalian and yeast sterol 14alpha-demethylases which belong to the CYP51 family. Therefore it is conceivable that the obtusifoliol 14alpha-demethylase from plants also belongs to the CYP51 family, the only P450 family so far known to be conserved across the phyla.

Carbon Monoxide↗
PubMed1996

Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus.

In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (; ). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.

Animals↗
PubMed1996

Intracellular distribution of Arf proteins in mammalian cells. Arf6 is uniquely localized to the plasma membrane.

Subcellular distributions of the five human Arf proteins were examined, using a set of isoform-specific polyclonal and a pan-Arf monoclonal antibodies. Subcellular fractionation of cultured mammalian cells allowed the demonstration that Arf6 is uniquely localized to the plasma membranes of Chinese hamster ovary cells. The plasma membrane distrubution was unaffected by either GTPgammaS (guanosine 5'-O-(3-thio)triphosphate) or brefeldin A, an activator and inhibitor of Arf activities, respectively. In contrast, Arf proteins 1, 3, 4, and 5 were predominantly cytosolic but could be recruited to a variety of intracellular membranes, but not plasma membranes, upon incubation in the presence of GTPgammaS. The GTPgammaS-promoted binding of the cytosolic Arf proteins to membranes was blocked by brefeldin A. The stable association of Arf6 with plasma membranes and the insensitivity of its localization to either GTPgammaS or brefeldin A revealed a clear distinction between Arf6 and the other Arf isoforms. Localization of Arf6 to the plasma membrane suggests a unique cellular role for this isoform at the plasma membrane, but failure to find endogenous Arf6 on endocytic structures, including clathrin-coated vesicles, appears inconsistent with the proposed role of Arf6 in assembly of coat structures or endosomes in transfected fibroblasts (1,2).

ADP-Ribosylation Factors↗
PubMed1996

Effects of acid phospholipids on ARF activities: potential roles in membrane traffic.

ADP-ribosylation factors are a family of approximately 21 kDa GTP binding proteins which have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in mammals and yeast. Reversible membrane associations are thought to be an essential component in the physiological actions of ARF and are regulated by GTP binding. ARFs are unique among the superfamily of GTP binding proteins in having a strict dependence on phospholipids for nucleotide exchange. In addition, ARF proteins were found to bind phospatidylinositol 4,5-bisphosphate (PIP2) specifically. PIP2 was found to increase the rate of GDP dissociation and stabilize the nucleotide-free form of the protein. The previously described requirements for PIP2 in the ARF stimulated phospholipase D (PLD) activity and ARF GTPase activating protein (ARF GAP) assays provide the basis for a model in which PIP2 acts as a cofactor in one or more ARF pathways. There are potentially two distinct phospholipid binding sites each of which are coupled to the nucleotide binding site of ARFs.

ADP-Ribosylation Factors↗
PubMed1996

Human ARF4 expression rescues sec7 mutant yeast cells.

Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.

ADP-Ribosylation Factor 1↗
PubMed1995

ADP-ribosylation factor translocation correlates with potentiation of GTP gamma S-stimulated phospholipase D activity in membrane fractions of HL-60 cells.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors from both the membrane and the cytosol. The small GTP-binding protein ADP-ribosylation factor (ARF) has been established as one important component of PLD activation. By stimulating HL-60 cells with various agonists and then isolating the membrane fraction and assaying PLD activity in the presence and absence of GTP gamma S, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTP gamma S-stimulated PLD activity in the membrane fractions of these cells. Inactive phorbol esters induced no such potentiation. Both fMLP and active phorbol esters induced a 2-3-fold increase in GTP gamma S-stimulated PLD in HL-60 membranes. Membranes derived from stimulated HL-60 cells contained 60-70% more ARF as compared with membranes derived from control cells. Membrane contents of ARF were assessed by Western blotting with the anti-ARF monoclonal antibody 1D9 followed by densitometric evaluation. Therefore, ARF translocation correlates with the potentiation of the GTP gamma S-stimulated PLD activity. The effect on PLD activity and ARF membrane content achieved through fMLP stimulation was greatly enhanced by prior treatment of the cells with cytochalasin B. Membranes derived from control and fMLP-stimulated cells were assayed for PLD activity in the presence of exogenous ARF and a 50-kDa fraction known to contain elements implicated in PLD activation. The ability of ARF and the 50-kDa fraction to enhance GTP gamma S-sensitive PLD activity was significantly reduced when the membranes were derived from fMLP-stimulated cells. The data indicate that, in addition to ARF, elements of the 50-kDa PLD-inducing factors were likely already translocated to the membranes upon stimulation. We propose that ARF, upon stimulation with agonists such as fMLP or phorbol esters, is translocated to the membrane and in concert with other protein components of the 50-kDa fraction enhances PLD activity.

ADP-Ribosylation Factors↗
PubMed1995

Partial purification and characterization of Arf-sensitive phospholipase D from porcine brain.

Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5'-O-(3-thiotriphosphate) and the small GTP-dependent protein, Arf. While this activity was readily apparent in membranes from HL60 cells, it was much lower or not observable in membranes from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sepharose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and characterized with respect to size, nucleotide specificity, and sensitivity to different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s20,w of 5.1 and a Stokes radius of 4.3 nm. These parameters indicate that the enzyme has an apparent molecular mass of 95,000 Da. Effective stimulation of the enriched enzyme was achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derived from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective. PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity. Additionally, the ability of recombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine PLD that has been purified about 5,000-10,000-fold is synergistically activated by Arf in combination with other cytosolic components that are described in the accompanying paper (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several interacting regulatory subunits.

ADP-Ribosylation Factors↗
PubMed1995

The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch.

ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTP gamma S and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain. When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTP gamma S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP gamma S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.

ADP-Ribosylation Factor 1↗
PubMed1995

Mutational analysis of Saccharomyces cerevisiae ARF1.

Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly2 or Asp26 resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp26 (homologous to Gly12 of p21ras) was essential for the binding of the activating nucleotide, guanosine 5'-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21ras or Gs alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins. Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approximately 300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.

ADP-Ribosylation Factor 1↗
PubMed1995

Arf proteins: the membrane traffic police?

Cofactor for cholera toxin; activator of phospholipase D; regulator of coat-protein assembly; inhibitor of membrane traffic; ability to cause expansion of the endoplasmic reticulum and vesiculation of the Golgi; sensitivity to membrane phospholipids--each of these activities has been attributed to Arf proteins. Can a single molecular mechanism link them all?

ADP-Ribosylation Factors↗
PubMed1995

Nitric oxide modulates dopamine release during global temporary cerebral ischemia.

Dopamine (DA) is released in large quantities from the striatum during cerebral ischemia. Along with excitatory neurotransmitters, DA plays a role in cellular neuronal ischemic injury. In this study we examined the role of nitric oxide (NO) in the ischemia-induced release of DA. A microdialysis probe was stereotactically placed into the corpus striatum of 16 Sprague-Dawley rats for DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) level determinations. After probe stabilization, the animals received either NG-nitro-L-arginine-methyl ester (L-NAME), a NO synthase inhibitor, or vehicle through the microdialysis probe. Temporary global forebrain ischemia was induced using bilateral carotid artery ligature tightening and controlled hemorrhagic hypotension for 15 min. L-NAME administration caused a reduction in ischemic estimated extraneuronal DA concentration by 60% (P < 0.005) compared with control. There was an increase in both DOPAC and HVA concentrations during the recovery period compared to baseline values in the control group (P < 0.05). L-NAME also caused a reduction in HVA concentration compared to vehicle administration during the latter part of recovery (P < 0.05). These data support the concept that ischemic dopamine release may be mediated by NO. This NO-modulated DA release may contribute to the previously reported deleterious neurotoxic effects of NO during ischemia.

3,4-Dihydroxyphenylacetic Acid↗