Biomedical subjects

K J Gibson

Publications and source records attributed to K J Gibson.

At least 37 records · Page 2Linked to original sources

PubMed1997

Isolation and chemistry of the mixed anhydride intermediate in the reaction catalyzed by dethiobiotin synthetase.

Dethiobiotin synthetase (DTBS) catalyzes the formation of the cyclic urea, dethiobiotin (DTB), from (7R,8S)-diaminononanoic acid (DAPA), CO2, and ATP; the other products of the reaction are ADP and Pi. The first intermediate in the reaction sequence is the 7-carbamate of DAPA [Huang, W., et al. (1995) Biochemistry 34, 10985-10995; Gibson, K. J., et al. (1995) Biochemistry 34, 10976-10984; Alexeev, D., et al. (1995) Structure 3, 1207-1215]. The existence of the second postulated intermediate, a mixed carbamic-phosphoric anhydride formed when the carbamate is phosphorylated by ATP, is consistent with the cleavage of the gamma-phosphoryl group of ATP seen in DTBS reaction mixtures [Baxter, R. L., & Baxter, H. C. (1994) J. Chem. Soc., Chem. Commun., 759-760]. Two more direct lines of evidence for the mixed anhydride intermediate have now been obtained. First, a DTBS reaction mixture containing [18O]CO2 produced 18O-enriched DTB and Pi, as the existence of such an intermediate would require. Second, a moderately stable intermediate that could be labeled with either 14CO2, [gamma-33P]ATP, [9-3H]DAPA, or [1,7-14C]DAPA was trapped by quenching DTBS reactions at pH 4 and isolated by thin-layer chromatography. As expected for the proposed mixed anhydride, this species underwent acid hydrolysis to DAPA, CO2, and Pi; under basic conditions, the intermediate cyclized, yielding DTB and Pi. When returned to fresh enzyme at pH 7.5, the intermediate underwent cyclization at a rate comparable to that of normal turnover.

Adenosine Triphosphate↗

PubMed1997

Comparison of the rate-dependent properties of the class III antiarrhythmic agents azimilide (NE-10064) and E-4031: considerations on the mechanism of reverse rate-dependent action potential prolongation.

INTRODUCTION: Reverse rate-dependence, a lessening in Class III antiarrhythmic agent action potential duration (APD) prolongation as heart rate is increased, has been proposed to be related to an incomplete deactivation of the slow component (IKs) of the delayed rectifier K+ current (IK). The rate-dependent properties of block of IK by azimilide were compared to E-4031, which selectively blocks the rapid component (IKr) of IK, in guinea pig ventricular muscle. METHODS AND RESULTS: Azimilide prolonged APD in isolated papillary muscles in a concentration-dependent manner and to a greater degree than E-4031. Both agents prolonged APD less at fast than slow rates, consistent with a similar reverse rate-dependent effect. Isolation of azimilide block of IKs by subtraction of APD during E-4031 plus azimilide from E-4031 alone revealed rate-independent prolongation of APD. In voltage clamp experiments on single ventricular myocytes, activation of IKs was similar following 30 seconds of conditioning pulses of physiological duration (125 to 200 msec) with either a fast (cycle length 250 msec) or slow (cycle length 2000 msec) rate. The block of IKs by azimilide 3 microM was greater after a fast conditioning pulse train. CONCLUSIONS: Selective block of IKs prolongs APD in a rate-independent manner. In voltage clamped myocytes, no evidence of a rate-dependent accumulation of IKs was observed. These findings support a mechanism of reverse rate-dependent APD prolongation by Class III antiarrhythmic agents that block IKr independent of IKs.

Action Potentials↗

PubMed1997

Ovine fetal cardiovascular, renal, and fluid balance responses to 3 days of high arginine vasopressin levels.

To determine the effects of sustained high levels of arginine vasopressin (AVP) on the fetus and whether these effects were the same as those found during acute infusion of AVP, chronically catheterized fetal sheep aged 121-136 days were infused for 3 days with either AVP (45 mU x kg(-1) x h(-1)) or saline. The bradycardia, acidemia, and failure of glomerulotubular balance that occurred with acute AVP infusion were reversed by day 3 of AVP (P < 0.005) and the acute rise in arterial pressure was attenuated (P < 0.005). By contrast, the rise in the glomerular filtration rate was sustained (P < 0.005) and urinary osmolality increased further to 426 +/- 30 mosmol/kg (P < 0.01). Although placental blood flow did not change acutely with AVP, it had fallen by day 3 (P < 0.01). In addition, with AVP but not saline extracellular volume fell from 588 +/- 28 to 493 +/- 29 ml/kg (P < 0.002) and the plasma/interstitial volume ratio rose from 0.18 +/- 0.01 to 0.21 +/- 0.01 (P = 0.001). These findings suggest that although release of AVP may be beneficial in acute stress in utero, sustained high levels may be detrimental to fetal health and sodium balance.

Animals↗

PubMed1996

The effects of continuous drainage of fetal fluids on salt and water balance in fetal sheep.

1. In nine chronically catheterized fetuses in which all lung liquid was drained continuously from the time of surgery, the effects of continuous drainage of fetal urine for 1 week on fetal renal function, lung liquid production and salt and water balance were studied. Fetal wellbeing, as judged by fetal growth, urinary osmolality, blood gas status, arterial pressure and heart rate, was not adversely affected by urine drainage. The ewes, however, drank more water when fetal urine was drained. Thus. fetal plasma and urinary osmolalities declined (P < 0.25 and P < 0.05). 2. Fetal glomerular filtration rate fell from 75 +/- 4 ml kg-1 h-1 (+/- S.E.M., n = 9) before drainage to 54 +/- 7 ml kg-1 h-1 after drainage (n = 7; P < 0.005), and fetal renal sodium excretion also declined (P < 0.05). However, the excretion of sodium in lung liquid did not decrease and the fetal renin-angiotensin system was not activated. Fetal extracellular volume (561 +/- 44 ml kg-1, n = 7) and the calculated net sodium transfer (0.76 mmol kg-1 h-1, n = 6) and fluid transfer (15 +/- 2 ml kg-1 h-1, n = 8) to the fetus did not change. 3. It is concluded that overall fetal salt and water balance were maintained when all fetal urine and lung liquid were drained from fetal sheep in late gestation. Since drainage of urine and lung liquid considerably reduced the amniotic and allantoic fluids, transfer across the placenta and extraplacental membranes was able to compensate for the absence of these fluids. In response to the loss of sodium during drainage, fetal renal sodium conservation was about 11% of the total sodium conservation by the materno-fetal unit.

Animals↗

PubMed1996

Escherichia coli biotin synthase: an investigation into the factors required for its activity and its sulfur donor.

Biotin synthase catalyzes the chemically difficult final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. In the present work, we extend our characterization of this enzymatic reaction and the extensive set of factors required by it. A defined mixture of components that supports the biotin synthase reaction has been found. The mixture contains biotin synthase, flavodoxin, flavodoxin reductase, NADPH, Ado-Met, Fe, fructose-1,6-bisphosphate, cysteine, and dithiothreitol. Even though this defined mixture supports the biotin synthase reaction, and in that regard is an important step forward in the study of this enzyme, it is unlikely that it contains all the physiologically significant factors involved in the biotin synthase reaction since it supports as an upper limit the synthesis of only 2 mol of biotin per mole of biotin synthase monomer. Progress in our efforts to identify additional physiologically significant factors is also reported. First, we describe evidence that the fructose 1,6-bisphosphate in the defined reaction mixture is substituting for an unknown factor of considerably higher potency present in crude extracts. Second, we have found that a labile low-molecular-weight product of the 7,8-diaminopelargonic acid aminotransferase reaction stimulates the rate of biotin formation in the defined biotin synthase reaction mixture and can increase the final amount of biotin formed by threefold. This product seems to be derived from Ado-Met, which 7,8-diaminopelargonic acid aminotransferase uses as its amino donor. However, 5'-deoxy-5'-methylthioadenosine, the postulated breakdown product from the action of 7,8-diaminopelargonic acid aminotransferase on Ado-Met, cannot be the active material since it has no stimulatory effect when added to the biotin synthase reaction mixture. Third, with a defined reaction mixture in hand, [35S]cysteine and [35S]Ado-Met, two potential sulfur donors present in the defined reaction mixture, were tested separately as sulfur donors. No 35S was incorporated into newly formed biotin when either [35S]cysteine or [35S]Ado-Met was added to the defined biotin synthase reaction mixture.

Enzyme Activation↗

PubMed1996

Inhibition of IKs in guinea pig cardiac myocytes and guinea pig IsK channels by the chromanol 293B.

The chromanol derivative 293B was previously shown to inhibit a cAMP regulated K+ conductance in rat colon crypts. Subsequent studies on cloned K+ channels from the rat demonstrated that 293B blocks specifically IsK channels expressed in Xenopus oocytes, but does not affect the delayed and inward rectifier Kv1.1 and Kir2.1, respectively. In the present study, the specificity of 293B for the cardiac K+ conductances IKs and IKr, and for the cloned guinea pig IsK channel and the human HERG channel, which underly IKs and IKr, respectively, was analyzed. 293B inhibited both the slowly activating K+ conductance IKs in cardiac myocytes and guinea pig IsK channels expressed in Xenopus oocytes with a similar IC50 (2-6 micromol/l). In contrast, high concentrations of 293B had only a negligible effect on the more rapid activating IKr. Similarly, 293B exerted no effect on HERG channels expressed in Xenopus oocytes. In summary, 293B appears to be a rather specific inhibitor of IKs and the underlying IsK channels.

Animals↗

PubMed1996

Effects of losartan on the cardiovascular system, renal haemodynamics and function and lung liquid flow in fetal sheep.

1. The angiotensin type 1 (AT1) receptor antagonist, losartan (10 mg/kg) was infused intravenously into nine chronically catheterized fetal sheep (125-132 days gestation). Losartan reduced the fetal systolic (P <0.01) and diastolic (P <0.01) pressor response to 5 microg angiotensin II (AngII) i.v. from 27.4 +/- 1.5 to 7.4+/-0.9 and from 17.5 +/- 1.3 to 5.4 +/- 0.6 mmHg, respectively, after 1 h and to 6.1 +/- 0.5 and 4.4 +/- 0.5 mmHg, respectively, after 2 h. Maternal pressor responses to 5 microg AngII i.v. were unchanged. Fetal mean arterial pressure decreased (P <0.05) after losartan administration, but fetal heart rate did not change. 2. Fetal haematocrit increased (P <0.05), fetal PO2 decreased (P <0.01), PCO2 did not change and pH decreased (P <0.01), as did plasma bicarbonate levels (P <0.01) following administration of losartan. Thus, losartan induced a fetal metabolic acidosis. 3. Fetal placental blood flow did not change following administration of losartan. In the fetal kidney, losartan caused a decrease in vascular resistance (P <0.01) and an increase in blood flow (P <0.05). Glomerular filtration rate decreased (P <0.05); thus, filtration fraction decreased (P <0.01). There was no change in the fractional reabsorption of sodium and glomerulotubular balance was maintained. Free water clearance decreased (P <0.01) and became negative. Urine flow decreased (P <0.01), the excretion rates of sodium, potassium and chloride did not change, but the urinary sodium: potassium ratio decreased (P <0.05). There was a decrease in lung liquid flow (P <0.05) following losartan. 4. It is concluded that the fetal renin-angiotensin system (RAS) is important in the maintenance of fetal arterial pressure, the regulation of fetal renal blood flow and is essential in the maintenance of fetal glomerular function. Further, these actions of AngII are mediated via functional AT1 receptors. These effects of losartan on the fetal cardiovascular system, renal blood flow and function are similar to those observed following captopril administration. Thus, the effects of angiotensin converting enzyme (ACE) inhibition in the foetus are due to the blockade of the fetal RAS and are independent of any direct effects on bradykinin or prostaglandin levels.

Animals↗

PubMed1996

Hypotonic-induced stretch counteracts the efficacy of the class III antiarrhythmic agent E-4031 in guinea pig myocytes.

OBJECTIVES: The aim was to determine the effect and mechanisms by which myocyte stretch interacts with the prolongation of action potential duration (APD) by the class III antiarrhythmic agent E-4031. METHODS: Action potentials and whole-cell currents were measured in isolated guinea pig ventricular myocytes with a patch clamp procedure during perfusion of normotonic, normotonic with addition of E-4031, and hypotonic plus E-4031 solutions. RESULTS: Cell swelling leading to membrane stretch of myocytes in the whole-cell recording configuration occurred with hypotonic solution perfusion. APD, prolonged by E-4031, was reduced to less than control value with hypotonic-induced stretch. Evaluation of whole-cell currents after hypotonic-induced stretch revealed no significant changes in the L-type Ca2+ current, inward rectifier K+ current or the rapid component of the delayed rectifier K+ current. The slow component of the delayed rectifier K+ current (IKs) was upregulated and a stretch-induced CI- current was activated in hypotonic solutions. The hypotonic-induced modulation of these currents was not effected by protein kinase A or C inhibition. CONCLUSIONS: Hypotonic-induced stretch shortens APD and counteracts the effects of E-4031. This APD shortening is secondary to upregulation of IKs and activation of a stretch-induced Cl- current.

Action Potentials↗

PubMed1995

Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate.

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO2, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO2. However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. (1) Addition of diazomethane to mixtures of DAPA and [14C]CO2 yielded a mixture of the N7- and N8-methyl carbamate esters, consistent with carbamate formation in free solution. In the presence of excess DTBS (over DAPA), the ratio of N7:N8-methyl carbamate esters recovered was roughly doubled, suggesting that the enzyme preferentially bound the N7-DAPA-carbamate. (2) Both N7- and N8-DAPA-carbamates were observed directly by 1H and 13C NMR in solutions containing DAPA and [13C]CO2. In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. 13C NMR of mixtures containing enzyme, [7-15N]DAPA, and [13C]CO2 showed that the enzyme-bound carbamate was at N7 of DAPA. In addition, pulse-chase experiments showed that the binary complex of DTBS and N7-DAPA-carbamate became kinetically committed upon addition of MgATP. (3) The N7-DAPA-carbamate mimic, 3-(1-aminoethyl)nonanedioic acid, in which the carbamate nitrogen was replaced with a methylene group, cyclized to the corresponding lactam in the presence of DTBS and ATP; ADP and P(i) were also formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Diamino↗

PubMed1995

Mechanism of an ATP-dependent carboxylase, dethiobiotin synthetase, based on crystallographic studies of complexes with substrates and a reaction intermediate.

The crystal structures of six complexes of homodimeric Escherichia coli dethiobiotin synthetase with a variety of substrates, substrate analogs, and products have been determined to high resolution. These include (1) the binary complex of dethiobiotin synthetase and the N7-carbamate of 7,8-diaminononanoic acid, (2) the binary complex of enzyme and the alternate substrate, 3-(1-aminoethyl)-nonanedioic acid, (3) the binary complex of enzyme with the product ADP, (4) the quaternary complex of enzyme, ADP, the N7-carbamate of 7,8-diaminononanoic acid, and Ca2+, (5) the ternary complex of enzyme, the ATP analog adenylyl (beta, gamma-methylene)diphosphonate, and the N7-carbamate of 7,8-diaminononanoic acid, and (6) the quaternary complex of enzyme, the ATP analog adenylyl (beta, gamma-methylene)diphosphonate, 7,8-diaminononanoic acid, and Mn2+. One molecule of each substrate binds to one monomer of the enzyme. ADP and the ATP analogue bind to the classical mononucleotide binding fold with the phosphate groups close to the phosphate binding loop Gly8--Thr16 between beta-strand beta 1 and the N-terminus of alpha-helix alpha 1. The adenine ring is bound in a pocket between beta-strands beta 6 and beta 7. In the quaternary complex with Mn2+, the metal binding site is found in the vicinity of the beta- and gamma-phosphate groups. Two oxygen atoms from the phosphates and oxygen atoms from the side chains of Asp54, Thr16, and Glu115 are ligands to the Mn2+ ion in the quaternary complex. In the complex with ADP and the N7-carbamate of 7,8-diaminononanoic acid prepared in the presence of Ca2+ ions, a different metal binding site is found. The Ca2+ ion is coordinated to an oxygen atom of the alpha-phosphate group of the nucleotide, the side chain of Asp54, and solvent molecules. The 7,8-diaminononanoic acid substrate molecule interacts with residues from both subunits, making the dimer the minimal functional unit. The diamino group binds between the loops after beta 2 and beta 4, and the terminal carboxyl group at the hydrophobic tail of the substrate interacts with the amino terminus of helix alpha 5 and with the side chain of Tyr187 in helix alpha 6 of the second subunit at the monomer-monomer interface. Strong additional electron density close to the N7 nitrogen atom of the 7,8-diaminononanoic acid substrate in some complexes indicates that, even in the absence of added bicarbonate in the crystallization mixture, the carbamylated intermediate is formed in the crystal.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

PubMed1995

Extracellular volume and blood volume in chronically catheterized fetal sheep.

1. To determine the extracellular volume (ECV) in fetal sheep and its distribution between the plasma and interstitial spaces, ECV, blood volume (BV) and haematocrit were measured in ten chronically catheterized fetal sheep aged 121-133 days. Relationships with age, weight and other fetal variables, including glomerular filtration rate (GFR), were studied. 2. ECV was measured as the mean of the volumes of distribution of [3H]inulin and [14C]mannitol extrapolated to time zero. The time zero volume of distribution was 1506 +/- 79 ml (means +/- S.E.M.) for inulin and 1590 +/- 80 ml for mannitol. The ECV was 1548 +/- 79 ml (632 +/- 18 ml (kg fetal wt)-1). BV, measured using 51Cr-labelled red cells, was 351 +/- 27 ml (141 +/- 6 ml kg-1). Haematocrit, plasma volume and interstitial volume were 34 +/- 1%, 229 +/- 17 ml (92 +/- 3 ml kg-1) and 1319 +/- 63 ml (540 +/- 17 ml kg-1), respectively. 3. Interstitial volume per kilogram fell with increasing fetal weight (P = 0.026). BV per kilogram did not change with weight or age. 4. The plasma: interstitial volume ratio was 0.17 +/- 0.01. This ratio increased as fetal weight and age increased (P = 0.026 and P = 0.044), that is, the proportion of ECV that was contained outside the vascular compartment was lower in heavier or older fetuses. 5. Since GFR was 3.4 +/- 0.4 ml min-1, the entire fetal ECV was filtered by the fetal kidney only 3.1 +/- 0.3 times per day.

Animals↗

PubMed1995

Beta-adrenergic blocking property of dl-sotalol maintains class III efficacy in guinea pig ventricular muscle after isoproterenol.

BACKGROUND: Catecholamines antagonize the efficacy of several class III antiarrhythmic agents. To determine the role of the intrinsic beta-adrenergic blocking property of dl-sotalol in maintaining class III efficacy during a high-catecholamine state, we compared the electrophysiological properties of dl-sotalol with those of d-sotalol, which is devoid of significant beta-adrenergic blocking effect, before and after isoproterenol infusion. METHODS AND RESULTS: Action potential duration at 90% repolarization (APD90) was prolonged in isolated guinea pig papillary muscles perfused with d-sotalol and dl-sotalol 10(-4) mol/L over stimulation cycle lengths from 200 to 2000 ms. The increases in APD90 for d-sotalol and dl-sotalol over control were 10.9 +/- 2.5 to 23.7 +/- 4.8 ms and 27.9 +/- 4.0 to 39.0 +/- 5.6 ms, respectively. APD90 shortened to less than control in papillary muscles treated with d-sotalol but not dl-sotalol on addition of isoproterenol 10(-6) mol/L: -31.2 +/- 3.5 to -18.3 +/- 4.8 ms and 10.5 +/- 3.6 to 33.3 +/- 7.8 ms, respectively, P < .003. Single guinea pig ventricular myocytes were studied by the whole-cell patch clamp method. Time-dependent (Iout) and total (Itot) outward current in response to a 300-ms pulse to 20 mV and tail current (Itail) to -35 mV were measured after Ca2+ channel block and Na+ channel inactivation. Iout, Itail, and Itot were reduced in myocytes perfused with d-sotalol and dl-sotalol 10(-4) mol/L: Iout, -36.1 +/- 4.1%, -40.5 +/- 3.3%; Itail, -59.3 +/- 4.6%, -62.2 +/- 11.1%; Itot, -27.3 +/- 4.3%, -50.0 +/- 11.8%. Iout and Itot increased to a greater degree in myocytes treated with d-sotalol than dl-sotalol on addition of isoproterenol 10(-6) mol/L: Iout, 100.3 +/- 20.6%, 11.3 +/- 7.6%, P = .002; Itot, 86.8 +/- 39.2%, -41.1 +/- 20.9%, P = .01. Itail tended to increase more in myocytes treated with d-sotalol than dl-sotalol on addition of isoproterenol, but the difference was not significant (-9.1 +/- 13.5%, -28.0 +/- 9.0%). CONCLUSIONS: The beta-adrenergic blocking property of dl-sotalol maintains APD prolongation and repolarizing outward current block during isoproterenol infusion in guinea pig ventricular muscle. Extrapolation of these data to a clinical setting may explain the efficacy of dl-sotalol in diminishing ventricular arrhythmia recurrence.

Action Potentials↗

PubMed1995

Comparison of the transplacental transfer of enalapril, captopril and losartan in sheep.

1. The transplacental transfers of three drugs (enalapril, captopril and losartan) which block the renin angiotensin system and have different lipophilicities were studied in chronically catheterized foetal sheep (125-139 days gestation). 2. The ability of the foeto-placental unit to convert enalapril to enalaprilat was studied in two chronically catheterized foetuses. Enalapril (3 mg kg-1, 7.9 mumol kg-1) given i.v. to the foetuses abolished the foetal pressor response to 5 micrograms angiotensin I (AI) in one foetus and attenuated the pressor response in the other. 3. Enalapril (100 mg, 5.7 mumol kg-1) given i.v. to the ewe (n = 5) abolished the maternal pressor response to 2.5 micrograms AI (n = 1) and attenuated the maternal pressor response to 5 micrograms AI (n = 5, P < 0.001). The foetal pressor response to 5 micrograms AI (n = 2) and 10 micrograms AI (n = 3) did not change. The maternal and foetal pressor responses to angiotensin II (AII; n = 5) did not change. 4. Foetal pressor responses to 5 micrograms AI (n = 1) and 10 micrograms AI (n = 2) were attenuated within 11 min of their mothers (n = 3) being given i.v. captopril (15 mg, 1.5 mumol kg-1). Foetal pressor responses to 5 micrograms AII (n = 1) and to 10 micrograms AII (n = 2) did not change. 5. Losartan (100 mg, kg-1, 21.7 mumol kg-1) given i.v. to the foetus (n = 9) attenuated the foetal pressor response to 5 micrograms AII (P < 0.001) but the maternal pressor response to 5 micrograms AII did not change. 6. Losartan (100 mg, 21.7 MICROmol kg-1) given i.v. to the ewe (n = 5) attenuated the maternal pressor response to 5 microg AII (P <0.002) but the foetal pressor response to 5 microg AII did not change.7. It is concluded that the foeto-placental unit of the sheep can metabolize enalapril to enalaprilat.Captopril readily crosses the sheep placenta but enalapril and losartan do not. Thus, the transplacental transfer of these drugs does not parallel their lipid solubilities. Furthermore the results show that AT1 receptors are important in mediating the vasoconstrictor effects of AII in the foetus.

Angiotensin I↗

PubMed1994

Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 A resolution.

BACKGROUND: In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon. One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin. The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme. RESULTS: We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 A resolution. The subunit consists of a seven-stranded parallel beta-sheet, surrounded by alpha-helices. The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr. The mononucleotide binding part of the structure is very similar to the GTP-binding protein H-ras-p21 and thus all GTP-binding proteins. A comparison reveals that some of the residues, which in H-ras-p21 interact with the nucleotide and the metal ion, are conserved in the synthetase. CONCLUSIONS: The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold. Considerable similarities to the structure of the GTP-binding protein H-ras-p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold.

Adenosine Triphosphate↗

PubMed1994

Covalent attachment of palmitoleic acid (C16:1 delta 9) to proteins in Saccharomyces cerevisiae. Evidence for a third class of acylated proteins.

Saccharomyces cerevisiae was used as a model system to characterize the covalent attachment of palmitoleic acid to proteins. Chemically synthesized cis-[9,10-3H]hexadecenoic acid (palmitoleic acid) was used to demonstrate the attachment of this lipid species to at least six proteins (m = 122, 58, 45, 41, 31, and 17 kDa). The majority of the labeled proteins are distinct from those labeled with [3H]palmitic acid (16:0). Based on the lability of the bond in the presence of methanolic KOH or hydroxylamine (pH 8), we propose that [3H] palmitoleic acid is attached to proteins via a thioester linkage. The identity of the palmitoleic acid was established by C-18 reverse phase high performance thin layer chromatography and argentation thin layer chromatography analysis after the fatty acid was liberated from the proteins by either transesterification or saponification. Incorporation of [3H]palmitoleic acid into proteins was only slightly inhibited (relative to [3H] myristic acid) by the presence of cycloheximide, indicating that the attachment of [3H]palmitoleic acid occurs post-translationally. This report is the first description of multiprotein acylation by a long chain unsaturated fatty acid.

Acylation↗

PubMed1994

Levels of active and inactive reninlike enzymes in plasma and reproductive tract of the pregnant wallaby.

Active and total (acid-activated) levels of a reninlike enzyme (hereafter called renin) were measured in plasma, tissues, and yolk sac fluid of pregnant and postpartum wallabies. Plasma active renin generated angiotensin I (ANG I) from sheep angiotensinogen at 14 +/- 1.3 (SE) ng.ml-1.h-1, whereas acid-activated renin generated ANG I at 33.3 +/- 2.5 ng.ml-1.h-1, i.e., 44.2 +/- 3.7% of renin in plasma was active, and 58 +/- 3.7% was inactive. Inactive renin levels were highest in pregnant animals (P = 0.05). Uterine renin was mainly inactive (95%); levels were 5.1 +/- 1.1 times plasma levels. The levels of renin in nonpregnant uteri were the same as those in pregnant uteri from the same animals. Uterine renin levels did not change with gestation. Pooled acid-activated yolk sac fluid generated ANG I at low rates (0.7 and 1.6 ng.ml-1.h-1); the acid-activated supernatant of a homogenate of pooled fetal membranes generated ANG I at 15 ng.g wet wt-1.h-1. Yolk sac fluid was strikingly different in electrolyte composition from maternal plasma. Its lower osmolality suggests that the membranes separating it from maternal plasma have a low permeability to water. Thus, although eutherian and marsupial mammals diverged 136-164 million years ago, the wallaby, like many eutherian mammals, has inactive renin in blood, in the female reproductive tract, and in fetal membranes.

Animals↗

PubMed1994

Changes in renal function and blood volume in the newborn lamb delivered by cesarean section.

To determine the cause of the transient natriuresis in lambs within 1-2 h of birth, renal function and blood volume (BV) were measured in nine chronically catheterized fetal sheep aged 139-145 d before and after delivery by cesarean section. After delivery, sodium excretion increased 8-fold. This was due to a transient rise in glomerular filtration rate (by 39 +/- 21%, p < 0.02) and a fall in fractional reabsorption of sodium by the proximal tubule from 63.4 +/- 2.5% to 53.4 +/- 3.4% (p < 0.01). The distal tubule failed to compensate fully for this fall, because fractional reabsorption by the distal tubule rose from 35.5 +/- 2.4% to only 41.6 +/- 2.2% (p < 0.05). The extent of the natriuresis did not depend on the lamb's initial BV per kg at birth. However, the amount of fluid excreted and the clearance of sodium during a 45-min period within the first 1-1.25 h after birth were approximately equal to the fall in BV that occurred during this time. Thus, most of the fall in BV that occurs after delivery is due to renal salt and water losses. Because the natriuresis was greater if the lamb's arterial pressure rose after birth, it is possible that a high arterial pressure in the immediate newborn period could result in salt and volume depletion.

Animals↗

PubMed1993

Palmitoleate formation by soybean stearoyl-acyl carrier protein desaturase.

Palmitoyl (hexadecanoyl)-acyl carrier protein (ACP) was found to be an alternate substrate for recombinant soybean stearoyl (octadecanoyl)-ACP delta 9-desaturase. The fatty acid product was identified as palmitoleic acid ((Z)-hexadec-9-enoic acid), which suggests that the locus of desaturation was fixed with respect to the carboxyl, not methyl, end of each substrate acyl group. The apparent specificity factor (Vmax/Km) of the desaturase for stearoyl-ACP was > 100-fold larger than for palmitoyl-ACP, mostly because of the difference in Vmax for the two substrates.

Acyl Carrier Protein↗