Prostaglandin-E2-9-ketoreductase in rabbit kidney

The 100,000 x g supernatant of rabbit kidney contains a prostaglandin-E₂-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE₂ to PGF_2α. A broad pH profile was evident with an optimum at pH 7·5. Kinetic studies indicated a K_m of PGE₂. The isoelectric point was at pH 5·65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE₂-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney.

The 100,000 xg supernatant of rabbit kidney contains a prostaglandin-E2-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE2 to PGF2alpha. A broad pH profile was evident with an optimum at pH 7 with 5. Kinetic studies indicated a Km of 3 with 2 x 10-4M PGE2. The isoelectric point was at pH 5 with 65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE2-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Dual effects of bradykinin on prostaglandin metabolism: Relationship to the dissimilar vascular actions of kinins

Generation of a prostaglandin of the F series by bovine mesenteric veins in response to bradykinin may depend on increased synthesis of PGE and conversion of the latter to PGF after activation of PGE 9-ketoreductase by the kinin. The prostaglandin then mediates the constrictor action of bradykinin on the bovine mesenteric vein. A high speed supernatant (HSS) fraction of bovine mesenteric blood vessels contains the highest activity of PGE 9-ketoreductase. Incubation of PGE₂ with HSS at 37°C in the presence of a NADPH generating system resulted in time-dependent conversion of PGE₂ to PGF_2α. Bradykinin (0.01mM) more than doubled conversion of PGE₂ to PGF_2α by the PGE 9-ketoreductase obtained from mesenteric veins whereas the kinin had little effect on enzymic activity of the HSS fraction of mesenteric arteries. However, after inhibition of kininase catabolism, bradykinin increased PGE 9-ketoreductase activity of arteries and veins to the same degree.Prostaglandin release from veins by bradykinin appears essential to contraction of mesenteric venous strips evoked by the polypeptide as indomethacin treatment abolished this effect. PGE 9-ketoreductase may be an important prostaglandin regulatory mechanism of the vascular wall whereby the functional consequences of changes in rates of prostaglandin synthesis are governed by determining the ratio of PGE to PGF within vascular tissue. Constriction of bovine mesenteric veins evoked by bradykinin may, therefore, depend on increased prostaglandin synthesis and conversion of newly formed PGE to PGF, both steps being affected by the kinin.     

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2α

Antibodies directed toward PGF_2β were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium borohydride-reduced (³H) PGE₂ anti-PGF_2β binding by several prostaglandins. The antibodies to PGF_2β recognize the β-hydroxyl configuration in the cyclopentane ring of PGF_2β. With the use of both anti-PGF_2α and anti-PGF_2β, the product of PGE₂ reduction by 9-ketoreductase purified from chicken heart was identified as PGF_2α. Guinea pig liver and kidney homogenates were examined for PGE 9-ketoreductase activity. Although enzyme activity was present, no evidence of PGF_2β production was found.     

Modulation of prostaglandin of the renal vascular action of arginne vasopressin

To determine whether the renal vascular effect of arginine vasopressin (AVP) is modulated by renal prostaglandin E₂ (PGE₂) were determined during the infusion of AVP in dogs during control conditions and after the administration of the inhibitor of prostaglandin synthesis, indomethacin. During control conditions, intrarenal administration for 10 min of a dose of AVP calculated to increase arterial renal plasma AVP concentration by 75 pg/ml produced a slight renal vasodilation (p<0.01) and an increase in renal venous plasma concentration of PGE₂. Renal venous PGE₂ concentration during control and AVP infusion averaged 33 ± 7 and 52 ± 12 pg/ml (p<0.05), respectively. After administration of indomethacin, the same dose of AVP induced renal vasoconstriction (p<0.05) and failed to enhance renal venous PGE₂ concentration (9 ± 1 to 8 ± 1 pg/ml). Intrarenal administration of 20 ng/kg. min of AVP for 10 min induced a marked renal vasoconstriction (p<0.01) and increased renal venous plasma PGE₂. Renal venous PGE₂ during control and AVP infusion averaged 31 ± 10 and 121 ± 31 pg/ml (p<0.01), respectively. Administration of the same dose of AVP following indomethacin produced a significantly greater and longer lasting renal vasoconstriction (p<0.01) and failed to increase renal venous plasma PGE₂ (10 ± 1 to 9 ± 1 pg/ml). These results indicate that a concentration of AVP comparable to that observed in several pathophysiological conditions induces a slight renal vasodilation which is mediated by renal prostaglandins. The results also indicate that higher doses of AVP induce renal vasoconstriction and that prostaglandin synthesis induced by AVP attenautes the renal vasoconstriction produced by this peptide.     

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E₂ 9-ketoreductase, which catalyzes the conversion of PGE₂ to PGF₂, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE₂, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min^–1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA₂, PGE₁, and 13,14-dihydro-15-keto PGF₂, but not that of PGD₂, 11-PGE₂, PGH₂, PGJ₂, or ¹²-PGJ₂. The reaction product formed from PGE₂ was identified as PGF₂, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE₂ was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE₂ 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF₂ in human brain.Special issue dedicated to Dr. Sidney Udenfriend.     

Inhibition of renal PGE2-9-ketoreductase by diuretics

The metabolism of PGE₂ by extracts of renal cortex is species dependent. In the rat PGE₂-15-hydroxydehydrogenase initiates metabolism whereas in the rabbit PGE₂-9-ketoreductase predominates. In man both mechanisms may operate. Each of the metabolic enzymes, which limits the vasodilator-diuretic actions of PGE₂, was inhibited by ethacrynic acid, furosemide and indomethacin. Some inhibition of PGE₂-9-ketoreductase was also observed with chlorthalidone, hydralazine and phentolamine but the thiazide diuretics and a number of other cardiovascular-active agents were without significant effect. We conclude that the inhibition of PGE₂-9-ketoreductase and PGE₂-15-hydroxydehydrogenase could contribute to the mechanism of action of the non-thiazide diuretics in man.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH – and temperature optima of 3.9 and 50°C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-α-1,4-polygalacturonase (exo-PG I). K_m and V_max for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U × mg⁻¹, respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.     

Interaction between prostaglandin E2 and leukotriene D4 on the excretion of electrolytes by the dog kidney in vivo

Recent experiments indicate that prostaglandin E₂ potentiates the vasodilatory properties of leukotrienes in the skin microcirculation. The present experiments were undertaken to study the effect of leukotriene D₄ and prostaglandin E₂ on renal hemodynamics and urinary electrolytes in the dog. Experiments were performed in three groups of anesthetized Mongrel dogs: the first group was studied under hydropenia, whereas the two remaining groups were studied during water diuresis with (Group 3) or without indomethacin (Group 2). LTD₄ (100ng/min) and PGE₂ (3ug/min) were infused in the left renal artery to minimize systemic effects of these compounds. LTD₄ alone failed to influence urinary sodium excretion in all 3 groups. In Group 1, urinary sodium increased from 77 ± 6 to 393 ± 74uEq/min during PGE₂, and further increased to 511 ± 52uEq/min during LTD₄ + PGE₂. No change occured in the contralateral right kidney. In this group, glomerular filtration as well as renal plasma flow were not statistically influenced. In Group 2, the same phenomenon was observed for urinary sodium. The combined infusion of LTD₄ + PGE₂ increased urinary sodium without significant changes in glomerular filtration and renal plasma flow. Finally, in Group 3, indomethacin was shown to reduce the natriuretic effects of LTD₄ and PGE₂: during PGE₂ alone, urinary sodium increased from 90 ± 14 to 260 ± 66uEq/min, and only rose from 80 ± 10 to 175 ± 19uEq/min during the combined infusion of LTD₄ and PGE₂. In groups 2 and 3, free water clearance was utilized as an index of sodium chloride reabsorption in the thick ascending limb: this parameter increased from 2.35 ± 0.25 to 4.70 ± 0.30ml/min, while urinary volume was increasing from 3.55 ± 0.25 to 10.05 ± 0.65ml/min, during LTD₄ + PGE₂. Indomethacin, administered in Group 3, (3mg/kg/hr) again abolished the effect of combined PGE₂ + LTD₄. These results indicate a potentiating effect of leukotriene D₄ on the PGE₂-induced natriuresis in the anesthetized dog. These phenomena occured in the absence of significant changes in renal hemodynamics, therefore suggesting a direct tubular effect of these arachidonic acid metabolites. Finally, the water diuresis experiments suggest a proximal site of action of PGE₂ and LTD₄.     

Physico-chemical and Catalytic Properties of Clostridium perfringens Hyaluronidase: An Update

Hyaluronidase (E.C. 4.2.2.1 hyaluronate lyase) or Mu toxin is one of the main components ofClostridium perfringens toxin complex. Although this enzyme has been studied for many years, data on its physico-chemical and catalytic characteristics are still quite contradictory and lack lucidity and completeness. In order to update knowledge of enzymatic properties of clostridial hyaluronidase, a chromatographically purified preparation from C. perfringens type A BP6K free of side phospholipase C (alpha toxin), neuraminidase (sialidase) and collagenase (kappa toxin) activities was obtained and characterized. The purification procedure included the following steps: processing the culture liquid with calcium phosphate gel, precipitation of the enzyme with acetone, ultrafiltration, and chromatography on Sephadex G-100 column. The purified hyaluronidase was homogenous as judged by rechromatography, SDS-PAGE and isoelectric focusing. Being a glycoprotein, the enzyme was most active at pH 5.7–6.2 (depending on the nature of the buffer used), at temperatures 37–45°C and at a relatively high ionic strength (0.15 and higher). The hyaluronidase was unstable when at pH values below 5.0 and above 9.0 as well as at temperatures below 30°C and above 50°C. The enzyme was most sensitive to Cu²⁺, Pb²⁺and Al³⁺ions, while the inhibitory effect of EDTA was moderate. Molecular mass of hyaluronidase was 96kDa as estimated by gel filtration and 48kDa when estimated by SDS-PAGE, suggesting that enzyme is composed of two subunits. The isoelectric point of the enzyme was 4.4. Substrate specificity of the enzyme was narrow (appart from hyaluronate, it slightly split chondroitin, but did not split heparin and various chondroitinsulphates). Moreover, unsplit glycosaminoglycans appeared to be competitive inhibitors with K_ivalues 5.3×10⁻², 4.9×10⁻², 4.5×10⁻²and 4.2×10⁻²mg/mL for heparin, chondroitinsulphates A, B and C, respectively. The Michaelis constant in regard to potassium hyaluronate was calculated to be (15.4±2.6)×10⁻²mg/mL.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [³H] arachidonic acid ([³H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 μCi of [³H] AA and with 200 μg radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [³H] AA to PGE₂, PGF_2α, PGFM, 6-keto-PGF_1α, and to unidentified compounds in three chromatographic regions. One of these regions (region 1) contained triglycerides. Endometrial slices metabolized only small amounts of the [³H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF_2α (32.1 ± 17.9), PGFM (8.4 ± 6.2), PGE₂ (12.3 ± 7.5) and 6-keto-PGF_1α (41.4± 4.8), whereas day 16 conceptuses released more (P<.05) PGF_2α (9.0 ± 4.1) and 6-keto-PGF_1α (15.9 ± 2.7) than PGE₂ (0.9 ± 0.2) or PGFM (0.5 ± 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P<.05) PGFM (3.0 ± 0.2) and 6-keto-PGF_1α (4.0 ± 0.4) than PGF_2α (0.5 ± 0.08) or PGE₂ (0.05 ± 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [³H] AA to determine the effects of indomethacin on [³H] AA metabolism. In general, indomethacin (Id; 4 × 10⁻⁴ M) reduced (P<.05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [³H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [³H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embroynal survival and development, were synthesized in vitro by ovine conceptuses.     

A radioimmunoassay for 6-keto-prostaglandin F1α utilizing an antiserum against 6-methoxime-prostaglandin F1α: Application to study renal synthesis of prostacyclin

PGI₂ and 6-keto-PGF_1α were converted to 6-methoxime-PGF_1α (6-MeON-PGF_1α) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF_1α was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF_1α-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (³H-6-MeON-PGF_1α, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE₂, 1% with PGF_2α and less than 0.2% with PGD₂, PGF_1α, PGF_2β and TXB₂. The radioimmunoassay was used to estimate release of PGI₂ and 6-keto-PGF_1α from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf_1α radioimmunoassay was validated in biological samples by mass fragmentography. The chopped medulla (n=5) released 38±9 ng/g/min and the cortex (n=5) 4.7±2.0 ng/g/min, while the release of immunoreactive PGE₂ (iPGE₂) and iPGF_2α was 171±26 and 74±13 ng/g/min from the medulla and 4.3±1.3 and 2.7±0.3 ng/g/min from the cortex, respectively. The results confirm previous findings, which indicate that in the renal medulla prostaglandin endoperoxides are mainly transformed to prostaglandins, while in the cortex transformation to PGI₂ seems to be of greater importance.     

Prostaglandin specific binding in hamster myometrial low speed supernatant

A charcoal adsorption method was developed to measure specific prostaglandin binding in low speed supernates of hamster myometrial homogenates. This method was used to characterize and quantitate PGE₁-specific binding. The equilibrium binding constants and the concentration of specific PGE₁ binding sites were determined during the hamster estrous cycle. The apparent association constant for 12 different preparations was 1.16 ± 0.08 × 10⁹M⁻¹. The concentration of PGE₁ specific binding sites was significantly higher on Days 2 and 3 of the estrous cycle than it was on Days 1 or 4. The competition for PGE₁ binding sites by PGE₂, PGF_2α, PGA₁ and various PGE₁ metabolites and derivatives was measured in hamster myometrial homogenates. Relative affinities of the natural prostaglandins for the PGE₁ binding sites, calculated by parallel line assay, were: PGE₂>PGE₁>PGA₁>PGF_2α. For PGE₁ metabolites the relative affinities were: PGE₁>13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁>15-keto-PGE₁. For the analogs and derivatives the compounds tested ranked as: 16,16-dimethyl-PGE₁≥PGE₁>PGE₁ methyl ester>17-phenyl-18,19,20-trinor-PGE₁>15(S)15-methyl-PGE₁ methyl ester. Arachidonic acid, bis-homo-γ-linolenic acid and 7-oxa-13 prostynoic acid had relative affinities ≥0.1 compared to PGE₁=100. Indomethacin had a relative affinity of 0.4 compared to PGE₁.     

Modulation of human myometrial PGE2 receptor by GTP characterization of receptor subtype

We studied PGE₂ specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE₂ ≥ PGE₁ PGF_2α > Iloprost ≥ Carbacyclin ZK 110841 (PGD₂ analogue). These relative affinities indicated that the receptor was of the EP type.In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE₂ binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10⁻⁴ sec⁻¹ (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10⁻⁴ sec⁻¹ half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10⁻⁹ M to 4.96 ± 1.25.10⁻⁹M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10⁻⁴M. GppNHp and GTPγS were effective at 1.10⁻⁵M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE₂ binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla.Competition studies with PGE₂ analogues (sulprostone, 17-phenyl-ω-trinor PGE₂, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP₃ subtype.     

High-performance affinity capture-removal of bacterial pyrogen from solutions

Synthetic peptide S3Δ has high affinity for bacterial endotoxin or lipopolysaccharide (LPS). Under tested conditions of pH 5–9 and 0–0.4 M NaCl, the affinity constant, K_D ranged from 2·10⁻⁶ to 2·10⁻⁹ M⁻¹. A novel affinity matrix based on peptide S3Δ was developed for removal of LPS from solutions such as: water; buffers with a wide range of ionic strength and pH; medium for cell culture; and protein solutions under optimized conditions. At a starting LPS of ≈100 EU/ml, a post-purification level below 0.005 EU/ml was achieved.     

Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts

Metabolism of radiolabeled arachidonic acid (^*AA) by blastocysts and endometrial slices recovered from five gilts 16 days after detection of estrus was studies . Blastocysts from each gilt were divided into four 216 ± 18 mg, and each portion was placed into a separate petri dish containing 15 ml modified minimum essential medium (MEM)_. The incubates from each gilt received either 25, 50, 100 or 200 μg radioinert arachidonic acid (AA). Endometrium was dissected from each uterin horn, sliced and duplicate 509 ± 3 mg portions from each gilt were placed into petri dishes containing 15 ml MEM and 200 μm AA. All incubates received 5 νCi of ^*AA (either [¹⁴C]-arichidonic acid or [³H]-arichidonic acid). The incubates were rocked at 37°C for 24 h in an atmosphere of 50% n₂:45% O₂:5% CO₂. After incubation, tissues and MEM were separated by centrifugation. Metabolism of ^*AA was assessed in extracts of MEM and tissue homogenates by separating ^*AA and its metabolites on columns of Sephades LH-20. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α (^*PGFM), [³H]-PGE₂ (^*PGE₂) and [³H]-PGF_2^α (^*PGF_2^α). The greatest (P<.05) proportion (35.7 ± 1.8%) of the radioactivity in blastocyst MEM was recovered as PGE₂. In blastocyst homogenates, most (66.2 ± 3.3%; P<0.05) of the radioactivity was in a nonporal peak assumed to be arachidonate esters. The concentration of AA ni MEM did not alter metabolism of ^*AA by blastocysts. Endometrial slices produced ^*PGFM and ^*PGE₂ but only in small amounts, and they were capable of producing nonpolar, probably esterified, forms of ^*AA. It was concluded that porcine blastocysts produced and metabolized prostaglandins and that they make a contribution to the uterine milieu during early pregnancy.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Extracellular glucose-producing exodextranase of the yeast Lipomyces lipofer

A dextran-hydrolysing enzyme from Lipomyces lipofer IGC 4042 was purified from the supernatant of cultures grown on a mineral medium with dextran, by ultrafiltration and gel filtration on Bio Gel A-0.5 m. This preparation gave only one band by disc gel electrophoresis. Glucose was the only product of dextran hydrolysis. Optimum pH and temperature for the activity of the enzyme were pH 4.5–5.0 and 45°C, respectively. The enzyme was most stable over a pH range of 4.5–6.0, and after 2 hours at 50°C maintained over 60% of its original activity. The molecular weight was 29,000 daltons and the isoelectric point was at pH 7. K_m (45°C, pH 5) for dextran T-40 was 1.2×10^–5 M. Glucose inhibited the enzyme competitively with a K_i (45°C, pH 5) of 0.5 mM.     

Prostaglandin E2 secretion by day-6 to day-9 equine embryos

Prostaglandin E₂ (PGE₂) secreted by Day-6, Day-7, Day-8 and Day-9 equine embryos (ovulation = Day 0) during in vitro incubation was measured by radioimmunoassay. Embryonic PGE₂ secretion (ng/embryo/24 hr) was detectable on Day 6 (0.27±0.39), tended to increase (P <0.1) on Day 7 (0.57±0.88), and increased significantly (P <0.05) on Day 8 (2.23±0.86) and Day 9 (4.13±0.71). Embryo diameter at the start of the incubation period was linearly correlated (P <0.01) to embryonic PGE₂ secretion.