Novel prostaglandin dehydrogenase in rat skin.

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Synthesis of gamma-L-glutaminyl-[3,5-3H]4-hydroxybenzene and the study of reactions catalyzed by the tyrosinase of Agaricus bisporus.

gamma-L-Glutaminyl-[3,5-3H]4-hydroxybenzene was synthesized in order to study the kinetics of its hydroxylation by tyrosinase purified from Agaricus bisporus and to explore its role in the induction of the dormant state in the spores of this species. It was found to be unique among the monophenolic substrates for tyrosinase in that the lag period for the hydroxylation reaction decreased with increasing substrate concentration. Unlike previously studied compounds, this phenol appeared to function as an electron donor, allowing it to act as its own co-substrate in the hydroxylation reaction. Its catechol product, gamma-L-glutaminyl-3,4-dihydroxybenzene, was found to be a superior co-substrate, yielding its electrons more readily (oxidation peak potential +0.18 V as compared with +0.65 V for the phenol). In situ periodate oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone confirmed the co-substrate role of the catechol in the hydroxylation reaction. The tyrosinase-mediated oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone occurred with an apparent Km = 1.54 mM and Vmax = 0.36 mmol/min/mg of enzyme. gamma-L-Glutaminyl-4-hydroxybenzene acted as an inhibitor of the oxidation reaction.     

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.     

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Lead Enters Bovine Adrenal Medullary Cells Through Calcium Channels

Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 microM Pb2+, depolarization by 77 mM K increases the rate of Pb uptake from 12 +/- 1 to 47 +/- 5 mumol/(L cells X min). K-induced Pb uptake has an apparent Km for Pb2+ of 2.6 microM, and is antagonized by Ca2+ with a K0.5 of 1.4 mM. The Ca channel blocker D-600 inhibits Pb entry with a K0.5 of 0.4 microM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca.     

Purification and characterization of mevalonate kinase from suspension-cultured cells of Catharanthus roseus (L.) G. Don

Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G. Don suspension-cultured cells. The purified enzyme had an M(r) of 104,600 and a subunit size of about 41,500. Kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and ADP was the last product to leave the enzyme. True values for the kinetic constants were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM, and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was calculated to be 138.7 nkat/mg of protein. Product inhibition was only detectable at rather high concentrations: above 0.7 mM for 5-phosphomevalonate and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of 0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The pH-dependent activity profile of mevalonate kinase showed a broad pH optimum between pH 7 and 10, with a maximum at about pH 8.9.     

Thallous ion activation of p-nitrophenylphosphatase of rat myometrium plasma membrane

Addition of thallous ion (Tl+) inhibited the spontaneous mechanical activity of rat myometrium in K-free Krebs solutions with an I50 value of 30 microM. The corresponding value for I50 for similar inhibition by addition of K+ was 150 microM. Tl+ as well as K+-activated p-nitrophenylphosphatase (PNPPase) of isolated rat myometrium plasma membrane vesicles. Half maximal activation was caused by 0.47 mM Tl+ or 1.6 mM K+. Maximal enzyme activities obtained using Tl+ and K+ were comparable. The Km values for the substance p-nitrophenylphosphatase using Tl+ (1.24 mM) and K+ (1.46 mM) were also similar. Activation by either ion was inhibited by ouabain, Na+, inorganic phosphate, and vanadate (V +5). The results suggest that Tl+ can substitute for K+ for activation of the Na-K pump of rat myometrium plasma membrane.     

Heme synthetase in Trypanosoma cruzi

Heme-synthetase (Heme-S) has been studied in the epimastigote form of T. cruzi (Tulahuen and Y strains). The enzyme is confined to the "mitochondrial" fraction (sedimented at 30,000 g). Activity was dependent on protein and time of cell storage. Enzymic proto- and meso-heme formation was inhibited up to 40 and 72% respectively by Triton X-100. The optimum pH was 7.2 for protoheme and 7.5 for mesoheme formation. Heme-S reached its maximum when the concentration values were 37; 35 and 32 microM for proto-, meso-, and deuteroporphyrin, respectively. The activity is several times higher when mesoporphyrin is used as substrate. At a final conc of 100 microM Fe2+ and Zn2+ ions enhanced activity 200-400%. Cu2+ and Co2+ had no effect, while Mn2+ and Mg2+ were highly inhibitory. A combination of Fe2+ and Zn2+ at varying concentrations still showed great activation. However, at a fixed level of Fe2+, Cu2+ was changed into a strong inhibitor. We propose that, if it can be demonstrated that T. cruzi cannot multiply when protoheme is replaced by mesoheme, administration of mesoporphyrin would then greatly affect replication of T. cruzi. Furthermore, the addition of certain metals, such as Cu2+ to T. cruzi cultures might specifically inhibit the parasite enzyme opening the possibility of selectively destroying the hemoflagellate without affecting the host.     

Characterization of ferrochelatase in kidney and erythroleukemia cells

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.     

Uptake of γ-Aminobutyric Acid by Brain Tissue Preparations: A Reevaluation

The kinetic constants Km and Vmax for the uptake of gamma-aminobutyric acid (GABA) by various preparations from rat cerebral cortex were determined by means of Eadie-Hofstee plots and computer analysis. The Km values were much greater in 0.1-mm slices than in synaptosomal preparations, and the Km value increased further with the thickness of the slices. The apparent high Km values in slices were probably due to depletion of the GABA concentration in the extracellular fluid as the exogenous GABA ran the gauntlet of competing uptake sites on its way to sites deep within the slice, thereby bringing about a requirement for higher GABA concentrations in the incubation medium in order to maintain the internal GABA levels at the "Km level." Evidence was obtained for three GABA uptake systems with Km values (in synaptosomes) of 1.1 microM, 43 microM, and 3.9 mM, respectively. In contrast, only two uptake systems for D-aspartate were detected, with Km values of 1.8 microM and 1.8 mM, respectively. The implications of the findings in the study with respect to previous data in the literature are discussed.     

O-Acetylhomoserine sulfhydrylase of the fission yeast Schizosaccharomyces pombe: partial purification, characterization, and its probable role in homocysteine biosynthesis

A crude extract of Schizosaccharomyces pombe cells catalyzed sulfhydrylation of both O-acetyl-L-serine and O-acetyl-L-homoserine with H2S, but did not synthesize cystathionine from O-acetyl-L-homoserine and L-cysteine. The O-acetylhomoserine sulfhydrylase [EC 4.2.99.10] was very unstable; however, it could be stabilized by the addition of 25% (w/w) sucrose or glycerol. The optimal pH for activity was 8.0 and that for stability was 7.0. The enzyme was purified approximately 300-fold from an ammonium sulfate-precipitated fraction. L-Methionine was the most effective inhibitor among the amino acids examined. It inhibited the enzyme competitively with respect to OAH with a Ki value of 2.6 mM. Sulfhydrylase activity was inhibited to various extents by some carbonyl reagents, but sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid), and monoiodoacetic acid had no inhibitory effect. The enzyme also reacted with O-succinylhomoserine and L-homoserine to synthesize homocysteine directly, but could not utilize cysteine as a co-substrate in place of H2S. In the sulfhydrylation reactions, Km values for the substrates ranged from 10.4-12.5 mM. The enzyme was resolved to the apoenzyme by incubation with phenylhydrazine and reactivated by the addition of pyridoxal 5'-phosphate, whose Km value was 0.083 microM. The molecular weight of the enzyme was estimated to be approximately 186,000 by gel filtration and 170,000 by ultracentrifugation in sucrose density gradients. The isolectric point of the protein was pH 4.1. The characteristics of this enzyme are compared with those of physiologically functional sulfhydrylases reported for other organisms, and the possibility of the enzyme functioning as a homocysteine synthase is discussed.     

Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: Iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.     

Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115

Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.     

Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys

Three forms of N-acetyl-beta-D-glucosaminidase (NAG: A, B and I) were separated from baboon kidney using Con A-Sepharose and DEAE-Trisacryl chromatography. 2. The A form was further purified into two forms A-1 and A-2 using hydroxylapatite chromatography and anodic PAGE. Both were homogeneous on SDS-PAGE and anodic PAGE but microheterogeneous on PAG-IEF, which could be eliminated by prior treatment with endoglycosidase H or glycopeptidase F. 3. The carbohydrate content accounted for some of this microheterogeneity since it varied from 31 for A-1 to 17% for A-2 and the sialic acid was 6 and 1%. Deamidation may also contribute since the acidic amino acids (29 mol%) and ammonia were high following acid hydrolysis. 4. The mol. wt for A-1, determined by SDS-PAGE, was 52.1 K. 5. The pH optimum was 4.55 and the pI4.97. 6. The optimum temperature for NAG A and B was 50 degrees and 42 degrees C, but B retained more activity above 55 degrees C. 7. The Km for N-acetyl-beta-D-glucosamine and -galactosamine for both isoforms was 0.497 and 0.627 mM respectively. 8. Several ions were found to be uncompetitive inhibitors. Ag+ and Pb2+ were the most potent having Ki values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.     

Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.     

Purification and characterization of ferro- and cobalto-chelatases

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40,000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240,000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h, but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 microM for the metal and 30.3 microM for mesoporphyrin, and for cobaltochelatase activity, 27 and 45.5 microM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.     

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O2 half-saturation pressure, measured at 25 degrees C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 degrees C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -- deltalog p1/2 (O2)/deltapH = 0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve deltalogp1/2 (O2) = 0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O2 affinities at 25 degrees C which follows the order: proto less than meso less than deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with gparallel = 2.035 and by the low-field shift of gparallel. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with gparallel - 2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O2 affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in gparallel. The correlation of the cobalt hyperfine constants of the oxy forms with the O2 affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

Low-affinity potassium uptake system in the archaeon Methanobacterium thermoautotrophicum: overproduction of a 31-kilodalton membrane protein during growth on low-potassium medium.

During growth on low-K+ medium (1 mM K+), Methanobacterium thermoautotrophicum accumulated K+ up to concentration gradients ([K+]intracellular/[K+]extracellular) of 25,000- to 50,000-fold. At these gradients ([K+]extracellular of < 20 microM), growth ceased but could be reinitiated by the addition of K+ or Rb+. During K+ starvation, the levels of a protein with an apparent molecular weight of 31,000 increased about sixfold. The protein was associated with the membrane and could be extracted by detergents. Cell suspensions of M. thermoautotrophicum obtained after K+-limited growth catalyzed the transport of both K+ and Rb+ with apparent Km and Vmax values of 0.13 mM and 140 nmol/min/mg, respectively, for K+ and 3.4 mM and 140 nmol/min/mg, respectively, for Rb+. Rb+ competitively inhibited K+ uptake with an inhibitor constant of about 10 mM. Membranes of K+-starved cells did not exhibit K+-stimulated ATPase activity. Immunoblotting with antisera against Escherichia coli Kdp-ATPase did not reveal any specific cross-reactivity against membrane proteins of K+-starved cells. Cells of M. thermoautotrophicum grown at a high potassium concentration (50 mM) catalyzed K+ and Rb+ transport at similar apparent Km values (0.13 mM for K+ and 3.3 mM for Rb+) but at significantly lower apparent Vmax values (about 60 nmol/min/mg for both K+ and Rb+) compared with K+-starved cells. From these data, it is concluded that the archaeon M. thermoautotrophicum contains a low-affinity K+ uptake system which is overproduced during growth on low-K+ medium.