Isolation and properties of rat plasma lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.     

Change in Hatching Enzyme Activity during Development of the Sea Urchin, Hemicentrotus Pulcherrimus

The time course of change in hatching enzyme activity during development of embryos of the sea urchin Hemicentrotus pulcherrimus was observed. The enzyme was present in the particulate fraction in embryos until the time of hatching and was maximal at the time of hatching. Cell fractionation studies suggested the existence of an inhibitor of the hatching enzyme. This possibility was subsequently substantiated by experiments in mixtures of fractions: the activity of hatching enzyme in the particulate fraction was inhibited by the supernatant of embryos. This inhibitory factor was heat-stable and non-dialyzable, but it was not characterized further. The activity of secreted hatching enzyme was not inhibited by this factor, suggesting that the molecular forms of hatching enzyme in embryos and in the culture supernatant are different. After hatching, the amount of increase in the hatching enzyme activity in the culture supernatant was 3.5 times the amount of decrease in enzyme activity in the embryos, suggesting that the enzyme was activated during its secretion.     

Comparative study on human milk and serum biotinidase

Biotinidase was purified from human breast milk (4,000-fold), and was compared with human serum biotinidase (enriched 30,000-fold). The molecular weight of milk enzyme was 68,000 Da as determined by SDS-PAGE. It was definitely smaller than that of serum biotinidase (Mr = 76,000). Isoelectric point of milk biotinidase was 4.6, whereas that of serum biotinidase was 4.3. Sialic acid content in milk biotinidase was less than that found in serum enzyme. N-Acetyl-galactosamine was present in milk enzyme, whereas it was absent in serum enzyme. Milk biotinidase is O-glycosylated, whereas serum biotinidase is N-glycosylated. These differences in glycosylation suggest the existence of different types of excretion mechanisms between milk and serum biotinidase. Both biotinidases were found to be thiol-type enzyme, however, the extent of activation of the enzyme by 2-mercaptoethanol was 13-fold in milk, whilst the serum enzyme was activated only 1.5-fold. Km for biotinyl-4-amino-benzoate was 22 microM in milk enzyme and 50 microM in serum enzyme. Competitive inhibition by biotin (Ki) of milk enzyme was 43 microM and 1.3 mM for serum enzyme. These results suggest the structural differences at or near the active site of the each enzyme.     

Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids.

Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

Characterization of rat muscle fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase has been purified from rat muscle. Although the specific activity of the enzyme in the crude extract of rat muscle was extremely low, purification by the present procedure is highly reproducible. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of the muscle enzyme was 37,500 in contrast to 43,000 in the case of the liver enzyme. Immunoreactivity of the muscle enzyme to anti-muscle and anti-liver fructose 1,6-bisphosphatase sera was clearly distinct from that of the liver enzyme. All one-dimensional peptide mappings of the muscle enzyme with staphylococcal V8 protease, chymotrypsin, and papain showed different patterns from those of the liver enzyme. When incubated with subtilisin, the extent of activation of muscle fructose 1,6-bisphosphatase at pH 9.1 was smaller than that of the liver enzyme. The subtilisin digestion pattern of the muscle enzyme on SDS-polyacrylamide gel electrophoresis was distinct from that of the liver enzyme. The AMP-concentration giving 50% inhibition of the muscle enzyme was 0.54 microM, whereas that of the liver enzyme was 85 microM. The concentrations of fructose 2,6-bisphosphate that gave 50% inhibition of rat muscle and liver enzymes were 6.3 and 1.5 microM, respectively. Fructose 1,6-bisphosphatase protein was not detected in soleus muscle by immunoelectroblotting with anti-muscle fructose 1,6-bisphosphatase serum.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

Amylase of the thermophilic actinomycete Thermomonospora vulgaris.

alpha-Amylase of the thermophilic actinomycete Thermomonospora vulgaris was partially purified. Maximal enzyme activity was obtained at 60degreeC and pH 6.0. KM value was l.4%. The effect of some metal salts on enzyme activity was studied. Enzyme activity was inhibited by by KCN, EDTA, and iodoacetate. Inhibition by EDTA was completely nullified by CaCl2, but the inhibition by iodoacetate was not overcome by 2-mercaptoethanol. Exposure of the enzyme to pH 7.0 and 9.0 for 2 hr. did not affect the enzyme, but exposure to pH 3.0 for few minutes completely inactivated the enzyme. Exposure of the enzyme to 60degreeC resulted in an appreciable inactivation and exposure to 80degreeC completely inactivated the enzyme. Addition of CaCl2, 2-mercaptoethanol, or enzyme substrate the 60degreeC exposed enzyme. However, bovine serym albumin had a protective effect when the enzyme was exposed to 60degreeC but not to 80degreeC. The enzyme was stable in the presence of 8 M urea.     

DISTRIBUTION AND PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME OF RAT BRAIN

Abstract— Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.     

Distribution of post-proline cleaving enzyme in human brain and the peripheral tissues

Summary We have studied the distribution of post-propline cleaving enzyme activity in the various tissues in humans using 7-(succinyl-Gly-Pro)-4-methylcoumarinamide as substrate. The post-propline cleaving enzyme activity was high in muscle, testes, kidney and submandibular gland, but was low in the heart, mesenterium and aorta. In the brain, relatively high post-propline cleaving enzyme activity was observed in the cerebral cortex, but other brain regions showed a very low enzyme activity.On Sephadex G-100 column chromatography, enzyme activity in human kidney showed a major peak and a minor peak. The major peak coincided with the enzyme in human cerebral cortex, but was different from human serum enzyme. Diisopropylfluorophosphate, a serine protease inhibitor, strongly inhibited the enzyme activity of each active fraction. The enzyme in the cerebral cortex and kidney was inhibited by heavy metals and thiol blocking agents. However, inhibition of enzyme activity in the serum was not observed with such inhibitors. Therefore, we suppose that post-proline cleaving enzyme activity in the brain is similar, if not identical, to that in the kidney.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Presence of two DNA polymerases in Tetrahymena pyriformis.

Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced.     

Purification of inactivated photoresponsive nitrile hydratase

Photoresponsive nitrile hydratase from Rhodococcus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately 60 kDa and consisted of 2 subunits each having molecular weight of 27.5 and 28 kDa. The enzyme also contained 2 iron atoms/enzyme as a cofactor. The enzyme was more stable in its inactivated form, rather than the activated during storage in the dark. The enzyme was most stable in the temperature region of 0-35 degrees C, and lost its activity above 40 degrees C. The enzyme was most stable in the pH region of 6-8. The optimum temperature and pH for the enzyme activity was 30 degrees C and 7.8, respectively. The enzyme showed wide substrate specificity, and most of the metal ions did not affect enzyme activity significantly. The absorption spectrum revealed the presence of some cofactor which changed form after photoirradiation.     

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.     

Findings of trigonelline demethylating enzyme activity in various organisms and some properties of the enzyme from hog liver

Trigonelline demethylating enzyme activity was found widely in animals, plants and microorganisms. Very high enzyme activity of this enzyme was detected in hog liver. Properties of the hog liver enzyme were investigated. Optimum pH for the enzymic reaction was observed at 8.5. The Km value for trigonelline was calculated at 2.77 mM. Addition of any cofactor is not required for the reaction The enzyme activity was inhibited by heavy metal ions. The reaction product was identified as nicotinic acid. Proposed enzyme reaction mechanism and the role of this enzyme in biosynthesis and metabolism of NAD are discussed.     

Identification of the phosphocarrier protein enzyme IIIgut: essential component of the glucitol phosphotransferase system in Salmonella typhimurium.

The phosphoenolpyruvate-dependent phosphorylation of glucitol has been shown to require four distinct proteins in Salmonella typhimurium: two general energy-coupling proteins, enzyme I and HPr, and two glucitol-specific proteins, enzyme IIgut and enzyme IIIgut. The enzyme IIgut was solubilized from the membrane and purified about 100-fold, free of the other protein constituents of the phosphotransferase system. Enzyme IIIgut was found in both the soluble and the membrane fractions. The soluble enzyme IIIgut was purified to near homogeneity by gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on butylagarose. It was sensitive to parital inactivation by trypsin and N-ethylmaleimide, but was stable at 80 degrees C. The protein had an approximate molecular weight of 15,000. It was phosphorylated in the presence of phosphoenolpyruvate, enzyme I, and HPr, and this phosphoprotein was dephosphorylated in the presence of enzyme IIgut and glucitol. Antibodies were raised against enzyme IIIgut. Enzyme IIIglc and enzyme IIIgut exhibited no enzymatic or immunological cross-reactivity. Enzyme IIgut, enzyme IIIgut, and glucitol phosphate dehydrogenase activities were specifically induced by growth in the presence of glucitol. These results serve to characterize the glucitol-specific proteins of the phosphotransferase system in S. typhimurium.     

Reversible immobilization of invertase on Sepabeads coated with polyethyleneimine: optimization of the biocatalyst's stability

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 degrees C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 degrees C higher than that of soluble enzyme.     

A variant form of arylsulfatase A in human urine derived directly from the renal pelvis: kinetic and immunological characterization

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was examined in voided and in nephrostomic urine. A variant form of the enzyme was found in nephrostomic urine, in addition to the minor form, which is the sole component of arylsulfatase A in voided urine. The nephrostomic enzyme differed from the voided urine enzyme with respect to the kinetic parameters, the isoelectric point, heat stability and immunological reactivity. The isoelectric points of the voided urine and nephrostomic enzymes were 4.7 and 5.3, respectively. The nephrostomic enzyme was more heat-labile at 62.5 degrees C than the voided urine enzyme. Although the Km values of the two enzymes with nitrocatechol sulfate as substrate were almost the same, the V value of the nephrostomic enzyme was approx. one-hundredth that of the voided urine enzyme. The molecular weight (almost 130 000) did not differ between the voided urine and nephrostomic enzymes. It was demonstrated by various methods, using IgG antibody against the purified voided urine enzyme, that the nephrostomic enzyme was antigenically distinct from the voided urine enzyme.     

Potent fibrinolytic enzyme from a thermophilic Streptomyces megasporus strain SD5

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent broth of thermophilic Streptomyces megasporus strain SD5. The crude enzyme was concentrated using ammonium sulphate, dialysis and lyophilization. Approximately 0.11 mg ml(-1) crude enzyme with a specific activity of 4.2 U microg(-1) was obtained. Post-electrophoretic reactivity revealed a monomeric form of the enzyme with a molecular weight of 35 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 degrees C, respectively. The enzyme was resistant to a broad range of pH ranging from 6 to 9 and temperature ranging from 37 to 60 degrees C. The enzyme was a chymotrypsin-like serine peptidase and the activity of the enzyme was N-terminal-dependent. The in vitro clot lysis by the enzyme at 37 degrees C was encouraging.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.