Purification and properties of an extracellular lipase from the fungusBotrytis cinerea

An extracellular lipase (EC 3.1.1.3) from the fungusBotrytis cinerea has been purified to homogeneity and characterized. The purification included ammonium sulfate fractionation and sequential column chromatography. The purification of the preparation was 31-fold and recovery yield was 21%. The purified enzyme was associated with esterase activity according to activity staining on polyacrylamide gel. The molecular weight was determined as 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and estimated at 72 kDa using gel filtration, which suggests that the enzyme may be a monomer. The isoelectric point was 6.5, and optimal activity was obtained at 38°C and pH 6.0. This lipase showed a high specificity for synthetic substrates containing long-chain unsaturated fatty acids using umbelliferone esters. The effect of β-cyclodextrin on the hydrolysis of olive oil has been studied. The specific activity was 25 μmole/min/mg in the absence of β-cyclodextrin and 132 μmole/min/mg in its presence.     

Studies on linoleic acid 8R-dioxygenase and hydroperoxide isomerase of the fungusGaeumannomyces graminis

Linoleic acid is sequentially converted to7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungusGaeumannomyces graminis, which is a common pathogen of wheat. The objective of this study was to separate and characterize the two enzyme activities. The isomerase activity was found mainly in the microsomal fraction of the mycelia and the 8R-dioxygenase in the cytosol. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelectric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retention. Isoelectric focusing in the presence of ethylene glycol (20%) indicated an isoelectric point of pl 6.1–6.3. The enzyme was retained on strong anion exchange columns at pH 7.4 and could be eluted with 0.3–0.5 M NaCl. Incubation of the enzyme with 0.1 mM linoleic acid led to partial inactivation, which may indicate product inhibition. Paracetamol and the lipoxygenase inhibitor ICI 230,487 at 30 μM inhibited the 8R-dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadecadienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence ofp-hydroxymercuribenzoate. The hydroperoxide was rapidly converted by the hydroperoxide isomerase in the microsomal fractions to7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid. The isomerase was neither inhibited by carbon monoxide nor by ketoconazole (100 μM). The isomerase was active over a broad pH range. It could be separated from the 8R-dioxygenase by differential centrifugation, by ammonium sulfate precipitation and by gel filtration. We conclude that the linoleic acid 8R-dioxygenase and the hydroperoxide isomerase are two separate enzymes.     

Properties of diacylglycerol kinase purified from bovine brain

A nearly homogeneous but somewhat unstable diacylglycerol kinase (ca. MW 72,000 daltons) was purified from bovine brain by modification of the procedure of Kanoh et al. (Kanoh, H., Kondoh, H., and Ono, T. [1983]J. Biol. Chem. 258, 1767–1774). The purification consisted of four steps (brain cytosol isolation and successive chromatography on DEAE-cellulose, Sephadex G-25 for desalting and ATP-agarose) carried out in buffers stabilized with EDTA, ATP and dithiothreitol (DTT). Specific activities, determined within 4 hr of purification, ranged from 908–1857 nmol ATP incorporated/min/mg protein, with the variation reflecting the instability. Optimal activities required deoxycholate (0.1%), one of the phosphoglycerides [phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS)] (0.025–0.25 mM), ATP (5 mM, apparent K_m=0.57 mM), 1,2-dioleoyl-rac-glycerol (5 mM, apparent K_m=1 mM) and Mg²⁺ (10 mM, apparent K_m=2.2 mM). Phosphatidylinositol (PI) was slightly less effective than PC, PE or PS and noninhibitory in combination with PC, PE or PS. Relative to PC phosphatidic acid (PA) (52%), sphingomyelin (48%), lyso-PC (1.5%) and lyso-PI (28.6%) were less effective activators. The sulfhydryl reagents,p-chloromercuribenzoic acid (PCMB) (1.0 mM),N-ethylmaleimide (NEM) (1.0 and 2.0 mM) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (1.0 mM), showed strong inhibition of activity which was prevented by 0.5 mM DTT. In contrast to other reports, this purified enzyme showed no monoacylglycerol kinase activity. Comparison of diacylglycerols of varying fatty acid composition indicated that the enzyme showed a preference for substrates with at least one unsaturated fatty acid, particularly in the 2-position. With saturated fatty acids the order of preference was C₁₀ and C₁₂>C₁₄>C₁₆>C₁₈. Such a pattern indicates that the enzyme shows little selectivity that favors the generation of particular molecular species of PA.     

Partial purification and characterization of sphingosine N-acyltransferase (ceramide synthase) from bovine liver mitochondrion-rich fraction

Sphingosine N-acyltransferase (ceramide synthase, E.C. 2.3.1.24) was solubilized from bovine liver mitochondrion-rich fraction with n-ocytl β-d-thioglucoside as the detergent and partially purified by sequential chromatography on columns of DE-32, shingosine affinity, and Sepharose CL-6B. The partially purified preparation migrated on SDS-polyacrylamide gel electrophoresis as two major protein bands of 62 and 72 kDa. The molecular mass of the enzyme estimated by gel filtration was 240–260 kDa, suggesting that the partially purified enzyme is present in a subunit form or simply has an aggregative nature. The specific activity of the final preparation for the condensation of sphingosine with stearoyl-CoA increased by 98.7-fold compared with the starting material. The optimal pH value for the ceramide synthesis was 7.5. The partially purified enzyme had an apparent K _m of 146 μM and a V _max of 11.1 nmol/min/mg protein for stearoyl-CoA. The K _m and V _max values toward sphingosine were 171 μM and 11.3 nmol/min/mg protein, respectively. Interestingly, sphinganine was also a good substrate for this enzyme, and the K _m and V _max values were 144 μM and 8.5 nmol/min/mg protein, respectively.     

Specific limited hydrolysis and phosphorylation of food proteins for improvement of functional and nutritional properties

Limited specific hydrolysis of casein byStaphylococcus aureus V8 protease was used to produce 2% and 6.7% hydrolysates (2 and 6.7% of the peptide bonds hydrolyzed), each containing five polypeptides (by gel filtration) ranging in size from ∼ 16,000 to ∼1,000 daltons. The mixtures of polypeptides had substantially increased solubilities at pH 4.0–4.5, near the isoelectric point of casein. In general, the emulsifying activity index was less for the hydrolysates than for casein; the emulsion stability was higher for the 2% hydrolysate than was the emulsion from casein. Phosphorylation of zein markedly increased the water solubility of zein above and below pH 4. When the free amino acids tryptophan and/or lysine were added to zein in the presence of POC1₃, some amino acids were covalently bound to zein, in addition to covalent attachment of phosphate groups. Threonine did not become incorporated into zein by this method. These derivatives were much more soluble than zein above and below pH 4, the minimum solubility point. A derivative containing 0.98 mol P/mol of zein, along with 1.05% tryptophan and 0.24% lysine, had a relative growth effect onTetrahymena thermophili of 49% that of casein, in comparison to 4.5% for unmodified zein. All the modified zeins had improved emulsifying activity indices.     

Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver

Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (K_d=10⁻¹⁵ M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures—polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [¹⁴C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons ±3.3%. This protein was shown to bind avidin (M_r=16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [¹⁴C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein. The polyethylene glycol precipitation yielded 200 μg protein (4.4 μg/g liver), with a specific activity of 5 nmol malonyl-CoA/min/mg protein, whereas avidin affinity chromatography yielded 1.75±1.1 mg protein (9.0 μg/g liver), with a specific activity of 1.37±0.18 μmol malonyl-CoA/min/mg protein. The enzyme was citrate dependent showing maximum activity between 10 and 20 mM. Acetyl-CoA carboxylase-specific activity decreased by 50% in the presence of 0.2 M NaCl. These findings suggest that the major protein (M_r=230,000) purified from rainbow trout liver is acetyl-CoA carboxylase with enzyme characteristics comparable to mammalian acetyl-CoA carboxylase.     

Purification and properties of lipase from the anaerobepropionibacterium acidi-propionici

A lipase secreted by the anaerobePropionibacterium acidipropionici was purified 52-fold with 27% recovery by employing a three-step purification protocol. The enzyme has a small molecular mass (Mr = 6000–8000) as determined by gel filtration and ultracentrifugation. It hydrolyzed palm oil, coconut oil, castor oil, olive oil, groundnut oil and tributyrin. Enzyme activity was inhibited by Ni²⁺, Ba²⁺, Mg²⁺, Cu²⁺, ethylenediaminetetraacetic acid, iodoacetamide, N-acetylimidazole and nonidet P-40 but stimulated by Ca²⁺, Co²⁺, K⁺, Fe²⁺, sodium dodecyl sulfate and N-bromosuccinamide. The enzyme showed substrate inhibition for both tributyrin andp-nitrophenyl acetate.     

Inevitable generation of primary alcohols during reduction of oxidized lipids with sodium borohydride

This report deals with the fluorometric determination of fatty alcohols generated by the reduction of the ester linkage of lipids with NaBH₄, and with the limitations of the reduction method for assaying oxidized lipids. Optimum conditions for the fluorometric analysis of primary and secondary alcohols using 1-anthroyl nitrile were obtained. After reduction with NaBH₄ in MeOH or in MeOH/benzene (8∶2, v/v), the formation of 1-hexadecanol from a variety of palmitic acid esters was measured fluorometrically by reverse-phase high-performance liquid chromatography (HPLC): From glycerides and methyl palmitate, 1–3% (w/w) 1-hexadecanol was produced and a trace was produced from cholesteryl palmitate (10 min, 21°C). 1-Hexadecanol was never generated from palmitic acid. Although considerable improvement occurred with the choice of the solvent for the NaBH₄ reduction, the generation of primary alcohols from ester lipids usually seems inevitable.     

Purification and characterization of thermophilic and alkalophilic tributyrin esterase fromBacillus strain A30-1 (ATCC 53841)

An extracellular esterase (EC 3.1.1.1) from a thermophilicBacillus A30-1 (ATCC 53841) was purified 139-fold to homogeneity by sodium chloride (6 M) treatment, ammonium sulfate fractionation (30–80%) and phenyl-Sepharose CL-6B column chromatography. The native enzyme was a single polypeptide chain with a molecular weight of about 65,000 and an isoelectric point at pH 4.8. The optimum pH for esterase activity was 9.0, and its pH stability range was 5.0–10.5. The optimum temperature for its activity was 60°C. The esterase had a half-life of 28 h at 50°C, 20 h at 60°C and 16 h at 65°C. It showed the highest activity on tributyrin, with little or no activity toward long-chain (12–20 carbon) fatty acid esters. The enzyme displayed K_m and K_cat values of 0.357 mM and 8365/min, respectively, for tributyrin hydrolysis at pH 9.0 and 60°C. Cyclodextrin (α, β, and γ), Ca²⁺, Co²⁺, Mg²⁺ and Mn²⁺ enhanced the esterase activity, and Zn²⁺ and Fe²⁺ acted as inhibitors of the enzyme activity. The enzyme activity was not affected by ethylenediaminetetraacetic acid, p-chloromercuribenzoate andN-bromosuccinimide. This paper was presented in part at the 82nd Annual Meeting and Exposition of the American Oil Chemists’ Society, held May 12–15, 1991, in Chicago, Illinois.     

Characterization and Immobilization of Marine Algal 11-Lipoxygenase from Ulva fasciata

The lipoxygenase (LOX) of the marine green alga Ulva fasciata was purified and immobilized in order to improve the stability and reusability. The algal LOX was partially purified by fractionation with 35–55% saturation of ammonium sulfate and MacroPrep high Q anion exchange chromatography. The LOX was purified ten times using linoleic acid (C_18:2) or arachidonic acid (C_20:4) as substrate, the Michaelis constant (K _m) of LOX was 117.6, 31.3 μM, and maximum velocity (V _max) was 12.8, 23.3 μmol hydroperoxy fatty acid/min-mg protein, respectively. The algal LOX showed the highest activity towards C_18:4 followed by C_20:4, C_18:2 and methyl ester of C_18:4. LOX activity increased up to 10.5 times with increased concentration of Triton X-100 in the extraction medium reaching an optimum at 0.05%. Calcium chloride, glutathione and phenylmethylsulphonyl fluoride were found effective protectants to LOX during purification. Hydroperoxyeicosatetraenoic acid (HpETE) formed from arachidonic acid catalyzed by this purified algal LOX was reduced and identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE) by NP-HPLC and GC–MS. This algal 11-LOX was immobilized in alginate beads. The stability was sevenfold greater than that of the unbound lipoxygenase at 4 °C in 0.05 M Tris–HCl buffer (pH 7.5). This is the first report on immobilization of a marine algal lipoxygenase with a view to its potential role in seafood flavor formation.     

Partial purification and characterization of acylester hydrolase from Lupinus mutabilis

An alkaline acylester hydrolase was partially purified from germinated seeds of Lupinus mutabilis. Hydrolytic activity was absent in the crude extract of ungerminated lupine seed, but it increased and peaked at the fourth day in the germinating seedling. The purification scheme involved homogenization, centrifugation, acetone precipitation, anion exchange chromatography, pH precipitation, and hydrophobic interaction chromatography. The acylester hydrolase was purified 126-fold, and the overall activity yield was 10%. The molecular weight estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis was 60 kDa. The enzyme had an isoelectric point of 6.2 and showed maximal activity at pH 8.0. The enzyme showed good stability between pH 5.0 and 9.0. In the pH range 7.0–7.5, enzyme precipitation was observed. The enzyme was stable from 0 to 25°C for 5 h and at 45°C lost 50% of its activity in the same period of time. At higher temperatures, the enzyme showed low thermal stability. However, the highest initial activity was found to be at 45°C. Nonionic surfactants and cholic acid enhanced the activity of the enzyme. The activity was reduced by the addition of toluene and isooctane and increased by the addition of diethyl ether, acetonitrile, methanol, and pyridine. The activity was reduced by 37% in the presence of 1 mM Cu²⁺ ions. The enzyme-hydrolyzed triolein showing no positional specificity.     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible for the cleavage of the 13‐hydroperoxides of linoleic and linolenic acids

An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13‐hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo‐product, 13‐oxo‐9(Z),11(E)tridecadienoic acid (13‐OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13‐hydroperoxide derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13‐hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13‐OTA along with the 13‐ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13‐ and 9‐hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH₄)₂ SO₄ precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0.     

Temperature sensitivity of cholesteryl ester hydrolases in the rat testis

Cholesteryl ester hydrolase (CEH) (EC 3.1.1.13) activity was assayed in the 104,000×g supernatant (S104) of rat and mouse testes and livers at various temperatures between 27 C and 44 C. The CEH activity in the testis dropped from 44 pmol [4-¹⁴C] cholesteryl oleate hydrolyzed/hr/mg protein to 14 pmol hydrolyzed/hr/mg protein (a 68% decrease) between testicular and abdominal temperatures (32 C and 37 C, respectively) in the rat. This decrease in activity is essentially a reversible phenomenon. CEH from the testis S104 was stabilized in 10 mM EDTA and was purified by HPLC size exclusion. These steps did not alter the temperature effect previously noted. The temperature effect on the testicular CEH was demonstrated in vivo by assaying the enzyme following unilateral cryptorchidism. The HPLC purification yielded 3 peaks of CEH activity from the testicular S104. The 28,000 MW peak was found to be temperature insensitive while the 70,000 and 420,000 MW peaks were temperature labile. The liver CEH of both species remained relatively constant over the range 32—37 C. CEH is a potential regulator of both steroidogenesis and membrane composition in the testis and its temperature lability may suggest a unique regulatory mechanism responsible for impaired spermatogenesis seen with elevated testicular temperatures.     

Synthesis,purification and characterization of 7-ketocholesterol and epimeric 7-hydroxycholesterols

Various methods were assessed for the synthesis of 7-ketocholesterol and epimeric 7-hydroxycholesterols. Upon the oxidation of cholesteryl acetate with t-butyl chromate, the resulting ketosterol acetate crystallized from methanol consisted of about 25% unoxidized cholesteryl acetate. After the sterol acetates were hydrolyzed in an aqueous K₂CO₃ medium, preparative TLC was used to fractionate the ketone from cholesterol. Of all the reducing agents employed, only LiAlH₄ reduced completely the purified 7-ketocholesterol to 7-hydroxycholesterols without side reaction products and ketone contamination. Yields of 21.3 mg of 7α-hydroxycholesterol and 72.6 mg of 7β-hydroxycholesterol were obtained by preparative TLC of a diol mixture prepared by the LiAlH₄-reduction of 100 mg of 7-ketocholesterol. To accomplish the preparative TLC separation of diol bands without overlapping, a double development of a chromatoplate with ethyl ether-cyclohexane (90∶10, v/v) and ethyl ether was essential. Data on the melting point, optical rotation and infrared spectra, as well as TLC and GLC characteristics, were obtained for purified 7-ketocholesterol and epimeric 7-hydroxycholesterols.     

Purification of peroxidase from Amsonia orientalis by three-phase partitioning and its biochemical characterization

The present work describes the purification and characterization of peroxidase from the medicinal plant, Amsonia orientalis, for the first time. The activity recovery for peroxidase was 162% with 12.5-fold purification. Optimal purification parameters were 20% (w/v) (NH₄)₂SO₄ saturation at pH 6.0 and 25°C with 1.0:1.0 (v/v) ratio of crude extract to t-butanol ratio for 30 min. The molecular mass of the enzyme was found to be ca. 59 kDa. Peroxidase showed K_m values of 1.88 and 2.0 mM for pyrogallol and hydrogen peroxide, respectively. FeSO₄, CuSO₄, HgCl₂, MnSO₄ and MgSO₄ did not inhibit the enzyme activity.     

Properties of an acid cholesteryl ester hydrolase inhibitor from rat serum

The inhibitory effect of a protein isolated from rat serum on lysosomal acid cholesteryl ester hydrolase (acid CEH; EC.3.1.1.13) activity was studied. An inhibitor was purified from rat serum following ultracentrifugation and heat treatment using column chromatography on Sephacryl S-200 and ultrafiltration. The purified inhibitor appeared as a single protein band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was 28,000 Daltons as judged by gel filtration on Sephacryl S-200 and SDS-polyacrylamide gel electrophoresis. The purified inhibitor was shown to be apolipoprotein A-I (apo A-I), the major apolipoprotein of high-density lipoprotein (HDL), using immunoprecipitation with rat anti-apo A-I immunoglobulin (Ig)G. Inhibition of acid CEH activity by apo A-I was dependent on the concentration of apo A-I. The values of V_max obtained were similar with or without apo A-I. Apo A-I of various other mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. Other apolipoproteins, such as apo A-II and apo B, also showed inhibiting activity. On the other hand, apo A-I had no effect on the activity of other enzymes found in lysosomes, such as cathepsin D, β-glucuronidase and acid phosphatase. The results suggest that apolipoproteins may play a role in the regulation of hydrolysis of cholesteryl esters in lipoproteins, that have been transferred to the liver, and that the inhibition of acid CEH activity by apo A-I may be a characteristic of the lipid-binding protein or be due to changes of the lipid/water interface.     

Plasma,apolipoprotein A-I and A-II levels in hyperlipidemia

Some of the component moieties of high denisty lipoproteins (HDL) were analyzed in normal subjects and in patients with hyperlipidemia. Apoproteins A-I and A-II were quantified by radioimmunoassay, HDL cholesterol and triglycerides were assessed on heparin-MnCl₂ supernates of fasting plasmas. We found that HDL is enriched in triglycerides in all forms of hyperlipidemia, while the proportion of ApoA-II is unaltered and the proportion of ApoA-I is decreased. Thus, the composition of HDL is altered in hypertriglyceridemia. The molecular associations of ApoA-I and ApoA-II in plasma were also examined by assaying the apoprotein contents of plasma fractions prepared by ultracentrifugation and by gel filtration column chromatography. The ApoA-I contents of d<1.063 fraction increased in hyperlipidemia from <0.5% to ∼2%, but the ApoA-I contents of the d>1.21 fraction remained at <12% of total in plasmas with triglyceride levels <1500 mg/dl. d>1.21 ApoA-I rose to 23% in one plasma with a triglyceride level of >1700 mg/dl. On column chromatography, ApoA-I eluted with the lipoproteins and also in a fraction whose molecular weight (MW) appreared to be ∼50,000 daltons. The proportion of plasma ApoA-I which eluted in the 50,000 MW peak was positively correlated with plasma triglyceride levels, but at triglyceride levels of <1500 mg/dl, <20% of ApoA-I was in the 50,000 MW peak. Between levels of ∼2000 and 12,000 mg/dl, the percentage “50,000 M.W. ApoA-I” was 20–25%. The ApoA-II contents of d<1.063 fractions were also increased in hyperlipidemia, but >95% of ApoA-II was found in the HDL fractions in both normal and hyperlipidemic plasma both by column chromatography and ultracentrifugation. Thus, the molecular association of ApoA-I appears to be altered in hyperlipidemia.     

Purification of lipoxygenase from Chlorella: production of 9- and 13-hydroperoxide derivatives of linoleic acid

Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9-and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30–45 and 45–80% saturated (NH₄)₂SO₄ precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH₄)₂SO₄ precipitation (35–80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorilla is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.     

CDPcholine:1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100,n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.