Peroxidase and hydrogen peroxide detection by a bioenergized method

Peroxidase activity and hydrogen peroxide concentrations were measured by a chemiluminescent method based upon the reduction of peroxidase Compounds I and II by both eosin and EDTA. Eosin excess present in the reaction mixture was excited during the reaction to its fluorescent state. This bioenergized method allows calculation of peroxidase concentration in the range of 10^?12 to 10^?7m, and hydrogen peroxide concentration in the range of 10^?9 to 10^?5m. This approach has been applied to the estimation of peroxidase activity in human red cell membranes and hydrogen peroxide formation in the peroxidase-catalyzed oxidation of glutathione.     

The methanolysis of some derivatives of 2,3,4-tri-O-benzyl-α-D-glucopyranosyl bromide in the presence and absence of silver salts

In contrast to reactions with high concentration, reactions of several derivatives of 2,3,4-tri-0-benzyl-α-D-glucopyranosyl bromide with low concentrations of methanol gave mainly the α-D-glucosides regardless of the structure of the C-6 substituent. Methanolysis of the same α-D-glucosyl bromides or the corresponding chlorides in the presence of silver tetrafluoroborate or hexafluorophosphate at —78° gave mainly the β-D-glucosides. The use of these silver salts led to side reactions, particularly when the glucosyl halide had an acyl blocking group at C-6. The side reactions were minimized when silver trifluoromethanesulfonate (triflate) was used. The relative amounts of α- and β-D-glucosides produced in the presence of silver triflate depended on the structure of the C-6 substituent and the solvent polarity. A rapid methanolysis of 2,3,4-tri-0-benzyl-6-0-(N-phenylcarbamoyl)-α-D-glucopyranosyl bromide with silver triflate in ether at —78° gave a high proportion of the methyl α-D-glucoside.The results of direct methanolysis seem to be due to competitive methanolysis of the anomeric bromides and a p?ush-pull$\text{\$}\text{?}$mechanism is postulated in the presence of silver tetrafluoroborate or hexafluorophosphate. Glucosyl triflate intermediates are proposed for the silver triflate-assisted methanolyses.     

Enzymatic isomerization (Δ7 → Δ8) of the nuclear double bond of 14α-alkyl substituted sterol precursord of cholesterol

The catalysis by rat liver microsomes under anaerobic conditions, of the conversion of [3α-³H]14α-methyl-5α-cholest-7-en-3β-ol and of [2,4-³H]14α-hydroxymethyl-5α-cholest-7-en-3β-ol to labeled 14α-methyl-5α-cholest-8-en-3β-ol and 14α-hydroxymethyl-5α-cholest-8-en-3β-ol, respectively, has been demonstrated. This finding is of importance in evaluating past research in this area and in consideration of pathways and mechanisms involved in enzymatic removal of carbon atom 32 of 14α-methyl sterols. Also described herein are syntheses of [2,4-³H]14α-hydroxymethyl-5α-cholest-7-en-3β-ol and 3β-acetoxy-14α-methyl-5α-cholest-8-ene.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

The photodynamic action of riboflavine on erythrocytes

The photodynamic action of riboflavine and riboflavine phosphate on erythrocytes was studied. It was found that irradiation of rat erythrocyte suspension in the presence of riboflavine phosphate resulted in an enhancement in erythrocyte potassium losses. Also, in the presence of riboflavine and light the release of lactose isonicotinoyl-hydrazone entrapped in released mouse erythrocytes, was markedly increased. The delayed photosensitizing effect, after keeping the irradiated samples for 20 hours in the dark, was more pronounced. No significant photohemolysis of erythrocytes could be demonstrated. Products of lipid peroxidation were formed, and was assumed to be responsible for the alteration in membrane permeability.     

Gymnemic acids: Secondary plant substances of dual defensive action?

Gymnemic acids, the triterpene saponins from the leaves of the asclepiad vine Gymnema sylvestre, act as feeding deterrents to a caterpillar, Prodenia eridania. The effect is demonstrable with sugar-free diets, suggesting that the acids do not act, as they do in mammals, by suppressing the sweetness of sugars.     

Fluorescence polarization assay of plasmin, plasminogen, and plasminogen activator

A chromatographic method involving medium-pressure liquid chromatography on alumina impregnated with silver nitrate is described for the separation of a series of closely related C₂₇ sterol precursors of cholesterol differing only in the number and location of olefinic double bonds. The features of the described system are compared with those of previously described thin-layer, gas-liquid, gravity column, and high-pressure liquid chromatographic methods.     

Inhibition of hepatic sterol synthesis and reduction of serum cholesterol in rats by 5α-cholest-8(14)-en-3β-ol-15-one

The preparation of 5α-cholest-8(14)-en-3β-ol-15-one from 3β-benzoyloxy-5α-cholest-8(14)-en-15-one is described herein. Subcutaneous administration of the former compound (2 mg per day for 15 days) resulted in a significant depression of the incorporation of the label of [2-¹⁴C]-acetate, but not of [2-¹⁴C]-3RS-mevalonate, into digitonin-precipitable sterols in rat liver homogenate preparations. Subcutaneous administration of the inhibitor, 2 mg per day or 5 mg per day, for 3 days resulted in a 12% and 22% reduction of serum cholesterol levels, respectively.     

Studies on the low temperature induced biogenesis of glycerol by adult Protophormia terranovae

Physiological and biochemical changes accompanying cold stress in the diapausing adult arctic blowfly, Protophormia terranovae, have been observed. In the laboratory, this insect survives prolonged periods at temperatures in the range of ?1°C to +4°C. Concentrations of free glycerol in excess of 10% of fresh body weight have been measured and the rate of its synthesis is greater at +1°C to +4°C than at ?1°C to 0°C. Under these conditions Protophormia also undergoes significant weight loss (up to 58% over 39 days) presumably in part due to dehydration. Its respiration rate decreases as expected when first shifted from 20°C to 0°C but the rate declines an additional 70% after exposure to 0°C for 24 hr. This lowest rate, which is then maintained, when considered with the initial faster one suggests positive thermal modulation is coupled to inverse thermal compensation during cold stress. This was not observed with nondiapausing Protophormia.Increments in free glycerol are accompanied by decreases in the insect's total glycogen reserves but upon rewarming, they return to pre-cold stress levels. While pre-stress glycogen stores are insufficient to provide for most of the free glycerol which accumulates, ingested carbohydrate present in the crop provides sufficient quantities. Studies with [¹⁴C] glucose indicate it is also metabolically active at low temperature.Neutral glyceride glycerol cannot contribute to net synthesis of free glycerol in significant amounts since the steady state concentrations present in pre-cold stressed insects decrease only slightly during cold stress. Furthermore, the specific radioactivity of acyl glyceride glycerol labelled in vivo with 2-[³H] glycerol before cold stress, remains unchanged during hibernation indicating that acyl glycerides are not turning over glycerol units produced by catabolism of hexose. The results of these studies argue that carbohydrate and not lipid glycerol is the source of the free glycerol which accumulates in Protophormia at low temperatures. The relationship of the above results to possible mechanisms which should permit glycerol accumulation under aerobic conditions are discussed.     

Synthesis and properties of some O-[2,2-bis(alkylthio)ethyl]-glycolaldehydes

O-[2,2-Bis(alkylthio)ethyl]glycoaldehydes (1a–e; alkyl = Et, Pr, Prⁱ, Bu^t, and -CH₂-, respectively) have been prepared from the corresponding O-[2,2-bis(alkylthio)ethyl]glycolaldehyde dimethyl acetals (2a–e) by acid hydrolysis. In anhydrous 1,4-dioxane in the presence of BF₃ · (Et₂O)₂,1a–c were partially transformed into glycolaldehyde bis(dialkyl dithioacetals),1d afforded trans-2,6-bis(tert-butylthio)-1,4-dioxane and 3,5-bis(tert-butylthio)-1,4-oxathiane, and1e did not react. The acetals2a–e) were prepared from the appropriate glycolaldehyde dialkyl dithioacetal by O-alkylation with bromoacetaldehyde dimethyl acetal.     

The formation of alpha-D-(1----3) branch linkages by a D-glucansucrase from Streptococcus mutans 6715 producing a soluble D-glucan

An exocellular D- glucansucrase that synthesizes a water-soluble, alpha-D-(1----6)-linked D-glucan having a high proportion of alpha-D-(1----3) branches was purified from the culture broth of Streptococcus mutans 6715. The rate of incorporation of D-[14C]glucose from [14C]sucrose into D-glucan of high molecular weight by this enzyme was increased (stimulated) by the presence of exogenous Leuconostoc mesenteroides B- 512F dextran, and it was found that this dextran could act as an acceptor. A highly branched dextran, containing 45-50% of alpha-D-(1----3) branch linkages, did not stimulate the enzyme nearly so much as B- 512F dextran, which has a low degree (5%) of alpha-D-(1----3) branches. We interpret this as evidence that the stimulating effects of dextran are not due to priming. If they were, the more highly branched dextran should have produced the greatest stimulation per unit weight, because a much greater number of nonreducing-end, priming sites would be available. We show that the D- glucansucrase was capable of transferring D-glucosyl groups from sucrose to B- 512F dextran to form alpha-D-(1----3) branches, thereby rendering the dextran more resistant to hydrolysis by endodextranase . The presence of 1.6M ammonium sulfate caused the enzyme to synthesize a D-glucan having a much higher percentage of alpha-D-(1----3) linkages.     

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.     

Functional-group modifications of dextran for linkage to a diazonium group. A potential vehicle for tumour targeting of antineoplastic triazenes

Several derivatives of dextran, containing a triazene side-chain, which are analogues of the antitumour agent DTIC (Dacarbazine), were prepared. The extent of triazene incorporation was measured by determination of the nitrogen content, and the presence of triazene groups was corroborated by u.v.- and n.m.r.-spectroscopic measurements. Some dextran-triazenes exhibited cytotoxic activity against M21 tumour cells in vitro.     

Relation between single-strand DNA mass and sedimentation distance in alkaline sucrose gradients

Single-strand breaks are introduced into bacteriophage T2 and Bacillus subtilis DNA in dilute solution with gamma rays and the DNA sedimented on alkaline sucrose gradients. Assuming (1) the number of single-strand breaks is linear with dose, and (2) the distance sedimented in alkaline sucrose gradients D is proportional to M^α (M is the single-strand DNA mass), the value of a is determined to be 0.40.     

Substrate stereochemistry of isovaleryl-CoA dehydrogenase elimination of the 2-pro-R hydrogen in biotin-deficient rats

(2R)-[³H]Isovaleric acid and (2S)-[³H]isovaleric acid (ammonium salts) have been synthesized. These substances, mixed with [1-¹⁴C]isovalerate, have been administered to biotin-deficient rats, which accumulate β-hydroxyisovaleric acid in their urine, the metabolite being formed via isovaleryl-CoA and β-methylcrotonyl-CoA. The results show that most of the tritium from (2R)-[³H]isovalerate was lost, and most of the tritium from (2S)-[³H]isovalerate retained in the conversion to β-hydroxyisovalerate. The stereochemistry of the isovaleryl-CoA dehydrogenase reaction is compared with the stereochemistry of other short-chain acyl-CoA dehydrogenase reactions.     

Alteration of processive mechanism of native polynucleotide phosphorylase by fixation to solid matrix.

Native $\text{E. coli}$ polynucleotide phosphorylase can be covalently bound to BrCN activated Sepharose. The Sepharose bound enzyme retains 70 % of its initial activity in polymerisation of nucleoside diphosphate. The Km of the enzyme for the polymerisation reaction in comparison to the soluble enzyme is not affected by its linkage to a solid matrix. The phosphorolysis of an hexanucleotide by the Sepharose-bound enzyme is not affected either. However, the rate of phosphorolysis of a long chain polynucleotide is dramatically altered. The Km values for poly(A) or poly(U) are increased by two orders of magnitude. The decrease of affinity for polymeric substrate is accompanied by a significant modification of the known processive mechanism characteristic of the native soluble enzyme.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.