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I H Segel

Publications and source records attributed to I H Segel.

At least 55 records · Page 3Linked to original sources
PubMed1981

Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism.

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...

Binding Sites↗
PubMed1979

Kinetic and chemical properties of ATP sulphurylase from Penicillin chrysogenum.

Adenosine triphosphate sulphurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4.) has been purified from the filamentous fungus. Penicillium chrysogenum, and characterized physically, kinetically, and chemically. The P. Chrysogenum enzyme is an octomer (mol. wt. 440 000) composed of eight identical subunits (mol. wt. 55 000). Some physical constants are S20,w = 13.0 X 10(-13)s, D20,w = 2.94 X 10(-7) cm2 X s-1, v = 0.733 cm3 X g-1, A1%1cm = 8.71 at 278 nm. The enzyme catalyses (a) the synthesis of adenosine 5'-phosphosulphate (APS) and MgPPi from MgATP and SO2-4, (b) the hydrolysis of MgATP to AMP and MgPPi in the absence of SO2-4, (c) Mg32PPi-MgATP exchange in the absence of SO2-4, (d) molybdolysis of MgATP to AMP and MgPPi, (e) synthesis of MgATP and SO2-4 from APS and MgPPi, and (f) Mg32PPi-MgATP exchange in the presence of SO2-4. The Vmax values of reactions (a)-(c) are about 0.10-0.35 mumole X min-1 X mg enzyme-1. The Vmax values of reactions (d)-(f) are about 12-19 mumole X min-1 X mg enzyme-1. The catalytic activity of the enzyme in the direction of APS synthesis is rather low (0.13 unit X mg protein-1, corresponding to an active site turnover number of 7.15 min-1). However, the ATP sulphurylase content of mycelium growing on excess SO2-4 is 0.22 unit X g dry wt.-1, which is sufficient to account for the maximum in vivo rate of SO2-4 assimilation. The normal catalytic reaction is Ordered Bi Bi with A = MgATP, B = SO2-4, P = MgPPi, and Q = APS. Several lines of kinetic evidence suggest that the E.MgATP and E.APS complexes isomerize (to E approximately AMP.MgPPi and E approximately AMP.SO4, respectively) before the second substrate binds. Chemical modification studies have disclosed the presence of essential arginine, histidine, carboxyl, and tryosine residues. The latter is rather acidic (pKa = 7 or less). Nitration of the tyrosine increases the Km for MgATP without significantly affecting Kia for MgATP or Vmaxf. This result, and the fact that MgATP plus nitrate protects the enzyme against inactivation by tetranitromethane while MgATP alone does not, suggests that the essential tyrosine plays a role in nucleotide isomerization (perhaps as an adenylyl acceptor).

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1979

ATP sulfurylase from Penicillium chrysogenum: is the internal level of the enzyme sufficient to account for the rate of sulfate utilization?

The in vivo rate of sulfate activation in Penicillium chrysogenum (wild-type strain ATCC 24791) was determined to be 0.19 +/- 0.09 mumol g(-1) (dry weight) min(-1) by the following methods. (i) The maximum growth of the organism in synthetic medium was a linear function of the initial Na(2)SO(4) concentration between 0 and 8 x 10(-4) Na(2)SO(4). The growth yield was 1.64 x 10(-2) g (dry weight) of mycelium per mumol of added sulfate, corresponding to a minimum sulfur requirement of 61 mumol/g (dry weight). Under these conditions (limiting sulfate) the minimum doubling time of P. chrysogenum in submerged culture was about 3.8 h, corresponding to a maximum exponential growth rate constant of 3.0 x 10(-3) min(-1). If all the sulfur in this mycelium passed through adenosine-5'-phosphosulfate, the rate of sulfate activation in vivo must have been 0.183 mumol min(-1) g(-1) (dry weight). (ii) In the presence of excess (35)SO(4) (2-), the total organic (35)S produced varied with the mycelial growth rate. However, until the culture approached maximum density, the product of [(growth rate constant) x (organic (35)S content)] was nearly constant at 0.24 to 0.28 mumol min(-1) g(-1) (dry weight). (iii) A sulfur-starved mycelium pulsed with 10(-4) M (35)SO(4) (2-) produced organic (35)S at a rate of about 0.10 mumol min(-1) g(-1) (dry weight) under conditions where the internal concentrations of ATP and sulfate would permit ATP sulfurylase to operate at about 70% of its V(max). Cell-free extracts of P. chrysogenum growing rapidly on excess sulfate contained 0.22 U of ATP sulfurylase per g (dry weight). Thus, in spite of the relatively low specific activity of homogeneous ATP sulfurylase (0.13 U/mg of protein, corresponding to an active site turnover of 7.15 min(-1)), the mycelial content of the enzyme was sufficient to account for the observed growth rate of the organism on inorganic sulfate as the sole sulfur source.

Biological Transport, Active↗
PubMed1979

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.

Adenosine Monophosphate↗
PubMed1978

Mechanism of the antimicrobial action of pyrithione: effects on membrane transport, ATP levels, and protein synthesis.

Pyrithione is a general inhibitor of membrane transport processes in fungi. A brief preincubation of Penicillium mycelia with pyrithione resulted in a marked decrease in the activities of a variety of independently regulated transport systems, including those for inorganic sulfate, inorganic phosphate, methylamine (actually, the NH(4) (+) permease), choline-O-sulfate, glucose, l-methionine (a specific system), and several hydrophobic l-alpha-amino acids (the general amino acid permease). The degree of inhibition at any fixed pyrithione concentration and exposure time increased as the pH of the incubation medium was decreased. This result strongly suggests that the active species is the un-ionized molecule and that pyrithione acts by collapsing a transmembrane DeltapH driving force. The degree of transport inhibition caused by a given concentration of pyrithione increased with increasing time of exposure to the inhibitor. However, exposure time and pyrithione concentration were not reciprocally related. At "low" pyrithione concentrations, transport inhibition plateaued at some finite value. This observation suggests that the fungi can detoxify low levels of the inhibitor. The concentration of pyrithione required for a given degree of growth inhibition increased as the experimental mycelial density increased. This phenomenon was consistent with the suggestion that the fungi are capable of inactivating pyrithione.

Adenosine Triphosphate↗
PubMed1976

Interaction of cyclic adenosine 3':5'-monophosphate with protein kinase. Equilibrium binding models.

A number of potential models for the interaction of cyclic AMP with protein kinase (RC or R2C2) have been examined. These include: Model 1, the simultaneous binding of cyclic AMP and release of C (catalytic subunit) from an independent RC protomer; Model 2, dissociation of an independent RC protomer prior to cyclic AMP binding to R (regulatory subunit); Model 3, cyclic AMP binding to RC prior to the dissociation of C; Model 4, random binding of cyclic AMP and dissociation of C with an interaction factor alpha less than 1; Model 5, release of 2C concomitant with the binding of one cyclic AMP to R2C2 followed by binding of the second cyclic AMP to the vacant R subunit; and Model 6, the simultaneous binding of cyclic AMP and release of C from one RC protomer resulting in a greater "affinity" of the other RC protomer for cyclic AMP, i.e., a cooperative version of Model 1. All the above models yield [cyclic AMP]0.5 values that increase with increasing protein concentration and Hill plots with average slopes equal to or less than 1.0 in the usual experimental range (10 to 90% of saturation). The Hill plots can be nonlinear, but for each model the exact shape of the plot changes in a characteristic (diagnostic) manner with changing protein concentration. Skeletal muscle protein kinase yields relatively linear Hill plots with napp values greater than 1.0. Consequently, Models 1 to 6 are not likely candidates. However, Model 2 is an excellent alternative model for proteins that display "negative cooperativity" with respect to the binding of a ligand. The properties of several "linear", "tetrahedral", and "all-or-nothing" cooperative models have also been examined. These include Models 7, A, B, and C and 8, A, B, and C which are cooperative versions of Models 2 and 3, respectively, and Model 9, a cooperative version of random Model 4. Model 9 is the most general model from which all others can be derived. Models 9 and 7, A, B, and C in which the prior dissociation of C greatly enhances or is an absolute requirement for cyclic AMP binding to R, are likely candidates for skeletal muscle protein kinase. All four of these models are capable of yielding Hill plots with average slopes greater than 1, and napp values that decrease with increasing protein concentration (in agreement with published data). In addition, in all four models the tight binding of MgATP to R2C2 yields decreased napp values and increased [cyclic AMP]0.5 values (also consistent with published data).

Binding Sites↗
PubMed1976

Adenosine triphosphate sulfurylase from penicillium chrysogenum. Steady state kinetics of the forward and reverse reactions.

The kinetic mechanism of ATP sulfurylase was established from initial velocity, product inhibition, and dead-end inhibition studies. In the forward direction, the reaction is steady state ordered, with MgATP=A, sulfate=B, MgPP1=P, and APS=Q.KmA=0.38 mM, Kia=0.71 mM, KmB=0.50 mM. Nitrate and chlorate are competive with sulfate and uncompetitive with MgATP. KiNO3-=0.25 mM; KiC1O3-= 0.15 mM. AMP and various MgATP analogs are competitive with MgATP and mixed-type inhibitors with respect to SO42-. The Ki for AMP is 0.55 mM. The reaction is rapid equilibrium ordered in the reverse direction with Kiq=0.3 to 1.0 muM and Kmp=0.65 muM. Adenosine 5'-phosphosulfate (APS) exhibits competitive substrate inhibition (KIQ=0.3 mM). The ratio Vmaxf/Vmaxr is 0.018. In the forward direction the ratio VmaxMoO42-/VmaxSO42- is 20. The Keq at pH 8.0 and 30 degrees calculated from the Haldane equation is 6 X 10(-9) to 3.3 X 10(-8) (depending on the Kiq value chosen). The experimental Keq is about 2.5 X 10(-9). The fact that Vmax/Vmaxr is about 1 million times greater than Keq is consistent with the assumed physiological role of the enzyme (APS synthesis). The mechanistic basis of the ordered binding sequence was probed by multiple inhibition analysis. Dead-end inhibitors competitive with MgATP (such as free ATP, Mg alpha,beta-methylene ATP, CrATP, and CaATP) do not induce substrate inhibition by sulfate or alter the inhibition patterns displayed by nitrate. This result suggests (but does not prove) that catalytic action on MgATP must precede the formation of the sulfate binding site.

Adenosine Triphosphate↗
PubMed1975

Kinetics of sulfate transport by Penicillium notatum. Interactions of sulfate, protons, and calcium.

The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.

Binding, Competitive↗