Apparent ornithine decarboxylase activity, measured by 14CO2 trapping, after frozen storage of rat tissue and rat tissue supernatants

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immobilization of aspartate aminotransferase on agarose

Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg/ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg/ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vmax was 51%. Chemical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 mg/ml of the gel). The apparent specific activities were 27-35%, while the value calculated from Vmax was 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vmax to 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined.     

Ontogeny and kinetics of carnitine palmitoyltransferase in liver and skeletal muscle of the domestic felid (Felis domestica)

The ontogeny of carnitine palmitoyltransferase (CPT) was examined in liver and muscle throughout growth and development of the domestic felid. Homogenates from animals in six age categories (newborn, 24-h, 3-, 6- and 9-week-old, and adult) were examined. Hepatic CPT specific activity increased progressively from birth to 6 weeks and then declined slightly into adulthood, with maximal values for animals greater than 24 h of age [171 nmol/(min g wet tissue)] being 70% higher than for newborns [99 nmol/(min g wet tissue)] (P<.05). Specific activity in adults was similar to that in 6- and 9-week-old juveniles. Total hepatic CPT activity [nmol/(min liver)] increased linearly with age, but the activity expressed per kg body weight [nmol/(min kg BW)] declined after 3 weeks. In contrast, skeletal muscle CPT-specific activity remained unchanged from birth to 3 weeks and then increased significantly, with maximal values at 9 weeks being 90% greater than those for young animals (newborn to 3 weeks; P<.05), whereas specific activity in adults was 50% lower than that observed in 9-week-old animals (P<.05). Hepatic and muscle apparent Km's for carnitine averaged 440 microM and did not vary with age. Hepatic carnitine concentrations remained relatively constant during development, but were lower in adult lactating females, whereas skeletal muscle concentrations increased markedly with age. Hepatic concentrations were 20-50% higher than apparent Km's for carnitine in young and growing animals, but concentrations were similar to the apparent Km at 6 weeks and significantly lower than the apparent Km in adults. Carnitine concentrations in skeletal muscle were 37% lower than apparent Km during the neonatal period, but significantly higher in cats >3 weeks of age. We conclude that postnatal increases in CPT activity support increased capacity for fatty acid oxidation in the developing felid and that dietary carnitine may be required to maximize enzyme activity.     

Expression of epithelial Na channels in Xenopus oocytes

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride- sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.     

Cofactor activity of dihydroflavin mononucleotide and tetrahydrobiopterin for murine epididymal indoleamine 2,3-dioxygenase

Dihydroflavin mononucleotide (FMNH2) and tetrahydrobiopterin (BH4) serve as cofactors for indoleamine 2,3-dioxygenase isolated from mouse epididymis. The optimal pH was between 7 and 8, and FMNH2-dependent activity was 4 to 5-fold higher than activity with methylene blue as the electron donor. Using FMNH2 with a FMN reductase system, the enzyme exhibited higher efficiency and specificity for L-Trp (an apparent Km of 1 X 10(-5)M and an apparent Vmax of 182 nmol/min/mg of protein). The apparent Km and Vmax for D-Trp were 6.2 X 10(-5)M and 31 nmole/min/mg, respectively. Consequently, these observations appear to present the first evidence for a flavin-dependent mammalian dioxygenase.     

Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity

A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, "leumorphin") to the tridecapeptide dynorphin B (Dyn B, "rimorphin"). This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent Km for the substrate of 0.58 microM. Surprisingly, a synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent Ki of 3.7 microM. The converting activity was also inhibited by Dyn A-(6-17) but not by Dyn A-(8-17), suggesting a role of Arg6-Arg7 in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition. Beta-Endorphin was an effective inhibitor of converting activity, and [alpha-N-acetyl]beta-endorphin was not, indicating a crucial role of the free NH2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent Ki of 39 nM. The converting activity was also inhibited substantially by ACTH-(1-13) but not by alpha-MSH, again indicating a requirement of the free NH2-terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings.

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 X 10(-5) M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 X 10(-4) M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 X 10(-4) M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a 'neutral' fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.     

Modification of the furanacryloyl-L-phenylalanylglycylglycine assay for determination of angiotensin-I-converting enzyme inhibitory activity

Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1) plays a central role in the regulation of blood pressure in man. The objective of this study was to evaluate and modify the furanacryloyl-L-phenylalanylglycylglycine (FAPGG) assay method for quantification of ACE activity. The fixed time conditions developed for assay of ACE activity were as follows: 0.8 mM FAPGG, 175 + or - 10 units l(-1) ACE, incubation at 37 degrees C for 30 min and enzyme inactivation with 100 mM ethylenediaminetetra-acetic acid (EDTA). Hydrolysis of FAPGG to FAP and GG was quantified by measuring the decrease in absorbance at 340 nm. It was shown that increasing the level ACE activity in the assay from 155 to 221 + or - 15 units l(-1) resulted in a corresponding increase in the apparent IC(50) value for Captopril from 9.10 to 39.40 nM. Similar trends in the apparent IC50 values for a whey protein hydrolysate were obtained. The results demonstrate the requirement for carefully controlling ACE activity levels in the assay in order to obtained comparable and reproducible values for the inhibitory potency of ACE inhibitors.     

Formylmethanofuran dehydrogenase from methanogenic bacteria, a molybdoenzyme

Formylmethanofuran dehydrogenase, a key enzyme of methanogenesis, was purified 100-fold from methanol grown Methanosarcina barkeri to apparent homogeneity and a specific activity of 34 mumol.min-1.mg protein-1. Molybdenum was found to co-migrate with the enzyme activity. The molybdenum content of purified preparations was 3-4 nmol per mg protein equal to 0.6-0.8 mol molybdenum per mol enzyme of apparent molecular mass 200 kDa. Evidence is presented that also formylmethanofuran dehydrogenase from H2/CO2 grown Methanobacterium thermoautotrophicum (strain Marburg) is a molybdoenzyme.     

Partial purification and properties of cGMP-dependent protein kinase from prawn tissue

Using ion-exchange chromatography and gel filtration, cGMP-dependent protein kinase was purified from prawn tissues 220-fold with a yield of activity of 12%. The apparent Ka values for cGMP, cAMP and 8-Br-cGMP are 1 . 10(-7), 5 . 10(-6) and 5 . 10(-8) M, respectively; the apparent Km values for ATP in the presence of cGMP is 9 . 10(-6) M. The cGMP-stimulated protein kinase activity was observed only in the presence of SH-compounds and high Mg2+ concentrations (500-100 mM). The protein kinase demonstrated a broad pH optimum wih a maximum at pH 6.8-7.2. The elution volume of the enzyme during gel filtration corresponded to a globular protein with molecular weight of 140,000.     

Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain

In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).     

Characterization of matrix metalloprotease activities induced in the sea urchin extraembryonic matrix, the hyaline layer.

Hyaline layers, freshly prepared from one-hour-old embryos, were devoid of gelatin-cleavage activity. However, upon storage at 4 degrees C, gelatin-cleavage activities appeared; three species of apparent mol mass 94 --> 117-, 90-, and 45-kDa were seen. All three species required zinc for activity. Using gel-exclusion chromatography we separated the 94 --> 117-, and 90-kDa species from the 45-kDa activity. The two higher mol mass species were inhibited by ethylenebis (oxyethylenenitrilo) tetraacetic acid and the lost activity was restored by calcium. Reconstitution of activity occurred with an apparent dissociation constant (calcium) of 5 microM. The presence of millimolar concentrations of magnesium had a minimal inhibitory effect on activity. The thermal denaturation profile of the higher mol mass gelatin-cleavage activity was significantly different in the presence and absence of calcium. Stabilization of these activities against thermal denaturation at 60 degrees C occurred with an apparent dissociation constant (calcium) of 0.6 mM. Magnesium had no significant effect on the thermal denaturation profile. Collectively, these results suggest at least two different modes of interaction between calcium and the higher mol mass gelatinases. These conclusions are discussed in the context of the high calcium and magnesium concentrations present in the sea water environment of the sea urchin embryo.     

Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells.

The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.     

Pyridoxine kinase in Plasmodium lophurae and duckling erythrocytes.

Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent Km for pyridoxine of the parasite enzyme was 6.6 times 10(-5) M whereas the host red cell enzyme Km was 1.9 times 10(-6) M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent Ki of 1.5 times 10(-6) and 8.6 times 10(-6) M, respectively. These results suggest that the vitamin B6 metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.     

Properties of adenylate cyclase from plasma membranes of the rabbit myometrium in different functional states

Adenylate cyclase of plasma membranes from the nonpregnant rabbit myometrium shows the maximum activity at pH 7.7-7.9, is characterized by apparent Km for ATP amounting to 0.38 +/- 0.09 mM, V--125 +/- 34.4 pmol min/mg protein, is activated at most by 15-20 mM Mg2+ and F-. Adenylate cyclase of plasma membranes from the pregnant rabbit myometrium is characterized by apparent Km for ATP amounting to 0.74 +/- 0.06 mM, V--77.3 +/- 6.0 pmol/min/mg protein, is activated at most by 5-10 mM Mg2+ and 10-15 mM F-; the pH optimum for the adenylate cyclase in this functional state is 7.3. Adenylate cyclase in the state of labour is characterized by apparent Km for ATP amounting to 0.46 +/- 0.11 mM, V--34.8 +/- 4.6 pmol/min/mg protein, is activated at most by 10-15 mM Mg2+ and F-, shows the same activity at pH 7.3-8.5. Adenylate cyclase of myometrium in three investigated states is activated by 2 mM EGTA; 10(-7) M Ca2+ decreases activation caused by EGTA; higher concentrations of Ca2+ decrease the basal activity of the enzyme.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Characterization of a calcium-dependent ATPase in Entamoeba invadens

A high-affinity calcium-dependent ATPase (Ca2+-ATPase) was identified in a crude plasma membrane fraction from Entamoeba invadens (IP-1 strain). The Ca2+-ATPase activity was solubilized from the membrane by utilizing the non-ionic detergent octylglucoside. The activity had an apparent half maximal saturation constant of 0.4 +/- 0.05 microM for free calcium. The calcium activation of ATPase activity followed a cooperative mechanism (Hill number of 2.3 +/- 0.13) which suggests that two interacting sites were involved. The high-affinity Ca2+-ATPase appeared to be magnesium-independent, since by lowering contaminant free magnesium with trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid did not modify the activity observed with Ca2+. The apparent Km of the enzyme for ATP was 31 microM. The observed activity had an optimum pH of 8.8. The enzyme was insensitive to various agents such as Na+, K+, ouabain, dicyclohexylcarbodiimide, KCN, NaN3, mersalyl, quercetin, ruthenium red and vanadate. Only lanthanum (0.5 mM) inhibited 100% the enzymatic activity. Calmodulin and trifluoperazine at the concentrations tested did not modify the Ca2+-ATPase activity.     

Buffer-dependent variations in assay of tyrosine aminotransferase holoenzyme

Homogenization of rat liver in Hepes (N-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid), MOPS (2-[N-morpholino]ethanesulfonic acid), Na phosphate, Pipes (piperazine-N,N′-bis[2-ethanesulfonic acid]), TEA (triethanolamine), TES (N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid), Tricine (N-tris-[hydroxymethyl]methylglycine), or Tris (tris[hydroxymethyl]aminomethane), and subsequent assay for supernatant total and holo tyrosine aminotransferase activity using these buffers yields apparent enzyme concentrations which vary depending upon the buffer composition, the ionic strength, and the fold-dilution of the supernatant. A precipitous decrease in the apparent holoenzyme concentration results from a slight dilution of the supernatant with most of the buffers. Some of the dilution effects may be due to dissociation of pyridoxal phosphate from the apoenzyme or to competition between the buffer and pyridoxal phosphate for association with the enzyme. The percentage of the apparent total enzyme which exists as holoenzyme varies from 3% for supernatant prepared in Na phosphate buffer up to 94% for that prepared in Hepes. Inactivation of total enzyme activity occurs to a similar extent resulting from incubation of liver homogenates prepared with Na phosphate, Hepes, or Pipes. The residual apparent holoenzyme activity observed when assayed in the presence of Na phosphate may be due to reaction of an enzyme other than tyrosine aminotransferase. The data provide a basis for explaining the large variation in reported percentage holoenzyme and should also serve as a warning for other holoenzyme assays which use pyridoxal phosphate as a cofactor.