Showing papers on "Enzyme assay published in 1986"
TL;DR: Experiments on rat liver cytosol have shown the specificity of the spectrophotometric method for superoxide dismutase, and common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations.
548 citations
TL;DR: A series of steroids were prepared and tested in vitro as inhibitors of human and rat prostatic 5 alpha-reductase and of binding of dihydrotestosterone to the rat androgen receptor and the primary structural modifications were changes of the A ring and of moieties attached at the C-17 position of the steroid nucleus.
Abstract: A series of steroids, primarily 4-azasteroids, were prepared and tested in vitro as inhibitors of human and rat prostatic 5 alpha-reductase and of binding of dihydrotestosterone to the rat androgen receptor. The primary structural modifications were changes of the A ring and of moieties attached at the C-17 position of the steroid nucleus. New A-ring modifications included the 4-cyano-3-oxo-delta 4 system in the carbocyclic series and 1 alpha-CN, 1 alpha-CH3, 1 alpha,2 alpha-CH2, 2 beta-F, 2-aza, 2-oxa, and A-homo changes in the 3-oxo-4-aza series. In addition, 4-azasteroids with a D-homo ring or methyl substitution at C-7 (alpha and beta) or C-16 (alpha and beta) were prepared. The majority of the C-17 substituents were prepared from reactive intermediates derived from the 17 beta-COOH. Enhanced 5 alpha-reductase inhibition in both the human and rat enzyme assays is seen with 4-CN substitution on 3-oxo-delta 4 steroids and with a C-17 side chain incorporating a lipophilically substituted semipolar group on the 4-aza-3-oxo-5 alpha-androstane nucleus. Fewer highly active compounds were found in the human enzyme assay than in the rat assay. Structural requirements for inhibition of the rat androgen receptor are much different from those for inhibition of the enzyme. The 17 beta-OH moiety enhances potency more than any other feature while introduction of double bonds at C-1 or C-5 in the azasteroid gives a small improvement. Azasteroids unsubstituted at the 4-position show greatly diminished receptor activity.
366 citations
TL;DR: Although the purified enzyme hydrolyzed some low-molecular-weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azOColl, bovine serum albumin, or gelatin, so this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine.
Abstract: An enzyme from Treponema denticola that hydrolyzes a synthetic trypsin substrate, N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), was purified to near homogeneity, as judged by gel electrophoresis. The molecular weight of the enzyme was estimated to be ca. 69,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ca. 50,000 by gel filtration on Sephadex G-100. The pH optimum for the hydrolysis of BAPNA was around 8.5. The enzyme was heat labile and irreversibly inactivated at low pH values. Enzyme activity was enhanced by Ca2+, Mg2+, and Ba2+ but inhibited by Mn2+, Hg2+, Co2+, and Zn2+. Metal chelators and sulfhydryl reagents had no effect on this activity. The enzyme was inhibited by certain protease inhibitors such as diisopropyl fluorophosphate, N-alpha-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethylchloromethyl ketone, alpha-1-antitrypsin, and soybean trypsin inhibitor. The Km values for BAPNA and N-alpha-benzoyl-L-arginine ethyl ester were 0.05 and 0.12 mM, respectively, and the Vmax values were higher than those observed with trypsin. Although the purified enzyme hydrolyzed some low-molecular-weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azocoll, bovine serum albumin, or gelatin. Thus, this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine.
224 citations
TL;DR: In Alzheimer's disease, the activities of both enzymes were increased, the glucose‐6‐phosphate dehydrogenase activity being almost double the activity of normal controls.
Abstract: The activities of the hexose monophosphate pathway enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were measured at autopsy in control and Alzheimer's disease brains. Enzyme activities did not vary between different areas of brain and were unaltered by age. In Alzheimer's disease, the activities of both enzymes were increased, the glucose-6-phosphate dehydrogenase activity being almost double the activity of normal controls. We propose that this increased enzyme activity is a response to elevated brain peroxide metabolism.
222 citations
TL;DR: The isolation of cDNA clones from HeLa cells, SV40-transformed human fibroblasts, human placenta and human teratocarcinoma cell lines have enabled us to sequence the entire coding region of Gd, providing the entire amino acid sequence of human G6PD for the first time.
Abstract: Glucose-6-phosphate dehydrogenase (G6PD) is an ubiquitous enzyme which by determining the NADPH level has a crucial role in NADPH-mediated reductive processes in all cells (1). The structural gene for G6PD, Gd, is X-linked in mammals and on the basis of its expression in many tissues, it can be regarded as a typical "housekeeping" gene (2). Over 300 variants of the protein are known, many of which have deficient enzyme activity. Nearly 100 of these variants are polymorphic in various populations (3). The mammalian enzyme is a homodimer or a homotetramer with a subunit molecular weight of approximately 56000 daltons (4). Here we report the isolation of cDNA clones from HeLa cells, SV40-transformed human fibroblasts, human placenta and human teratocarcinoma cell lines. These clones have enabled us to sequence the entire coding region of Gd. Thus, the entire amino acid sequence of human G6PD is provided for the first time. This work is the first step for structural analysis of G6PD variants and for an understanding of the biological features of this enzyme at the molecular level.
218 citations
TL;DR: The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity.
170 citations
TL;DR: The ability to examine GSHPx as a protein resulted in two new observations concerning the selenium-dependent GSHpx, which are that the plasma enzyme is antigenically distinct from the erythrocyte enzyme, and the second is that in the absence of seenium, there is a concomitant decrease in G SHPx protein.
161 citations
TL;DR: The role of inorganic nitrogen in the regulation of glutamate synthase activity was demonstrated in this paper, where it was shown that removing leaves and roots from NH4NO3-grown seedlings from a minus nitrogen medium for 24 hours reduced enzyme activity.
Abstract: Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 mM KNO3 or NH4Cl or 5 mM NH4NO3 had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH4NO3-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.
159 citations
TL;DR: It is concluded that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H(2)O(2)-mediated reaction cannot be ruled out.
Abstract: Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H2O2-mediated reaction cannot be ruled out.
157 citations
TL;DR: Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity, which imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetates or activated acetate.
Abstract: Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanoscarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 ..mu..mol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (M/sub r/ 250,000) formed aggregates with an M/sub r/ of approximately 1,000,000. The enzyme contained five subunits (M/sub r/s 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with /sup 61/Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate tomore » methane, possibly by cleavage of acetate or activated acetate.« less
154 citations
TL;DR: In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates, however, considerable differences between the bovines and human enzymes were observed with nucleoside substrates.
TL;DR: The subcellular distribution of ascorbate peroxidase and glutathione reductase in pea leaves was compared with that of organelle markers and isolated chloroplasts showed a close correlation between intactness and the percentage of enzyme activity recovered.
Abstract: The subcellular distribution of ascorbate peroxidase and glutathione reductase (EC 1.6.4.2) in pea leaves was compared with that of organelle markers. Enzyme distribution was found to be similar to that of the chloroplast enzyme NADPH-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). Isolated chloroplasts showed a close correlation between intactness and the percentage of enzyme activity recovered. Chloroplasts of 85% intactness were found to contain a high proportion of leaf dehydroascorbate reductase activity (EC 1.8.5.1), 10% of leaf glutathione and 30% of leaf ascorbate. These results are discussed in relation to the potential role of chloroplast antioxidant systems in plant resistance to environmental and other stress conditions.
TL;DR: It is demonstrated in the laboratory that vitamin E inhibits platelet phospholipase A2 in a dose-dependent manner and the importance of the hydroxyl moiety on the chromanol of the vitamin E molecule for its inhibitory action is emphasized.
TL;DR: Data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain and interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form ofThe enzyme in a calcium-dependent manner.
Journal Article•
TL;DR: The liver had the highest enzyme activity with minimal activity found in the kidneys, spleen, lung, colon, colon tumors, pancreas, breast tissue, breast tumors, bone marrow cells, and peripheral leukemic cells.
Abstract: Dihydrouracil dehydrogenase activity, with 5-fluorouracil used as the substrate, was measured in human tissues and leukemic cells. The liver had the highest enzyme activity (mean, 705 nmoles/g tissue/hr) with minimal activity found in the kidneys, spleen, lung, colon, colon tumors, pancreas, breast tissue, breast tumors, bone marrow cells, and peripheral leukemic cells. Wide variations in the enzyme activities were noted in samples collected from different subjects. 5-diazouracil inhibits the enzyme activity with the concentration required for inhibiting 50% (IC50) of the liver enzyme being 3 microM. Other compounds, thymine, thymidine, 6-methoxydiazouracil, and cyclo-5-diazouridine were also tested for their effect on this enzyme. With the exception of cyclo-5-diazouridine, all others produced inhibitory effect. The inhibitory effect of 6-methoxydiazouracil is similar to that of diazouracil. Thymidine and thymine are less active with identical values for IC50 of 80 microM.
TL;DR: The data suggest that human leukocyte 5‐LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.
TL;DR: Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography.
Abstract: Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. The enzyme is a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme does not appear to be a glycoprotein. It gives optimal activity with D-glucose, which is stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It is inhibited by Ag+ (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF, or KCN (each 10 mM).
TL;DR: Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized, revealing that serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatilserine biosynthesis in CHO cells.
TL;DR: Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme.
Abstract: The extractable activity ofL-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [(35)S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of(35)S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.
TL;DR: Aromatase cytochrome P-450 has been purified from human placenta to homogeneity, as demonstrated by electrophoresis on polyacrylamide gels with SDS, and by double diffusion against an antibody raised in rabbits.
TL;DR: A simple, sensitive fluorometric method for the determination of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) activity has been developed and should greatly facilitate the study of per oxisomal beta-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.
TL;DR: The enzyme activity changed in a manner suggesting its involvement in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.
TL;DR: An accurate assay of the rate-limiting enzyme in bile acid synthesis, the cholesterol 7 alpha-hydroxylase, in human liver is described, based on isotope dilution-mass spectrometry, and endogenous microsomal cholesterol is used as the only substrate for the enzyme.
Journal Article•
TL;DR: Differences in the cellular activities of the alkyl cleavage enzyme are not responsible for the differential cytotoxic responses between normal and specific types of neoplastic cells toward rac-1-octadecyl-2-methoxy-glycero-3-phosphocholine.
Abstract: The cytotoxic response of several types of neoplastic cells to analogues of unnatural alkyl phospholipids (e.g., rac-1-hexadecyl-2-methoxy-glycero-3-phosphocholine) has been partially attributed to their accumulation as a result of the low activity of the alkyl cleavage enzyme (a tetrahydropteridine-dependent monooxygenase) in tumor cells. We tested this possibility by comparing the alkyl cleavage enzyme activity in cells that exhibit differences in sensitivity toward the cytotoxic effects of the rac-1-hexadecyl-2-methoxy-glycero-3-phosphocholine. Human promyelocytic leukemia cells (HL-60), a cell line highly sensitive to the cytotoxic alkyl phospholipid analogue, possessed an alkyl cleavage enzyme activity (0.25 pmol/min/microgram protein) similar to that found in three cell types known to be relatively resistant to the cytotoxic activity of the analogue: immature human promyeloblastic leukemia cells (K562) (0.22 pmol/min/microgram protein), human polymorphonuclear neutrophils (0.34 pmol/min/microgram protein), and Madin-Darby canine kidney cells (0.37 pmol/min/microgram protein). Moreover, our results indicate that the cytotoxic rac-1-octadecyl-2-methoxy-glycero-3-phosphocholine analogue is not a substrate for the alkyl cleavage enzyme with an active microsomal preparation of the enzyme from rat liver; cleavage of this analogue was 200-fold less than the rate obtained with 1-octadecylglycerol as substrate. In cultures of either sensitive or resistant type cells, approximately 90% of the added rac-1-[9',10'-3H]octadecyl-2-methoxy-glycero-3-phosphocholine was not metabolized during a 24-h incubation. The amount of radiolabel in fatty acids, a major product of alkyl cleavage activity, was small, and essentially identical amounts were produced in all four cell types [3.1 +/- 0.2% (SD)]. These data indicate that differences in the cellular activities of the alkyl cleavage enzyme are not responsible for the differential cytotoxic responses between normal and specific types of neoplastic cells toward rac-1-octadecyl-2-methoxy-glycero-3-phosphocholine. On the other hand, the cellular uptake of the alkyl phospholipids could be a factor in explaining the cytotoxic response of certain tumor cells, since more radiolabeled 1-octadecyl-2-methoxy-glycero-3-phosphocholine was associated with the susceptible HL-60 cells than with the resistant cell types. Autoradiography revealed that the radiolabeled 2-methoxy analogue accumulates at the periphery of HL-60 leukemia cells, whereas the label was more uniformly distributed in polymorphonuclear neutrophils and K562 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: The mutated malate dehydrogenase gene showed resistance to substrate inhibition, an increase in both kcat and Km, and a shift toward a more acid optimum pH for the enzyme reaction.
TL;DR: In vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.
TL;DR: It was suggested that most lung cell antioxidant enzymes increased in activity following oxidant stress in proportion to increased cell mass, and the percent of whole lung enzyme activity accounted for by type II cells was calculated.
Abstract: The activity of antioxidant enzymes were measured in alveolar type II cells isolated from control and 85% oxygen-exposed rats to determine if type II cells, an oxygen resistant lung cell type had constitutively high enzyme activities and to measure the effect of hyperoxia on these antioxidant enzymes. Type II cells were isolated from lungs of control rats and rats exposed to 85% O2 for 7 days. In -whole lungs of rats exposed to 85% oxygen there is an increase in activity (per lung or per mg lung DNA) in the antioxidant enzymes CuZn superoxide dismutase, Mn super-oxide dismutase, catalase, glutathione peroxidase and glucose-6-phosphate dehy-drogenase. Oxygen exposure significantly increased (p < 0.05) all type II cell antioxidant enzyme activities when expressed per mg DNA. The protein content of oxygen exposed type II cells increased 25% from (63.9 ± 4.8 μg/106 cells to 79.6 ± 4.2 μg/106 cells, p < 0.05). When type II cell enzyme activities were expressed in U/mg cell protein, only CuZn superoxide dismuta...
TL;DR: This method permits the preparation of large amounts of stable, efficient homogeneous and well-defined substrate that is suitable for measuring the enzyme activity in plasma fractions.
Abstract: Publisher Summary Lecithin–cholesterol acyltransferase (LCAT) is synthesized by the liver and secreted into the bloodstream, where it is believed to act principally on the surface of high-density lipoproteins (HDL). The first attempts to isolate LCAT yielded only partially purified preparations even though a variety of chromatographic techniques were used, including hydroxylapatite adsorption, salt precipitation, ultracentrifugation, and affinity chromatography. Lecithin–cholesterol acyltransferase has been one of the most difficult enzymes to characterize from the physical, chemical, and enzymological points of view. The painstakingly slow early development of an efficient purification procedure and the instability of the enzyme precluded rapid progress. For the assay of LCAT activity, a stable, efficient, and uniform substrate that can be added to the enzyme source under conditions such that the concentration of substrate is not rate limiting is required. This method permits the preparation of large amounts of stable, efficient homogeneous and well-defined substrate that is suitable for measuring the enzyme activity in plasma fractions.
01 Jan 1986
TL;DR: Study of its biosynthesis, membrane orientation, and turnover in several tissues and cultured cell lines has broadened the authors' knowledge about Golgi function itself and in patients suffering from ovarian and breast cancer, increased levels of GT enzyme activity have been reported.
Abstract: Galactosyltransferase (GT) belongs to the glycosyltransferases. In several tissues and cell lines, the enzyme is localized by immunocytochemistry to the two to three trans cisternae of the Golgi complex and may thus be considered a specific membrane component of this type of endomembrane. As a consequence, it is the most common Golgi "marker" enzyme in cell fractionation studies. Study of its biosynthesis, membrane orientation, and turnover in several tissues and cultured cell lines has broadened our knowledge about Golgi function itself. The enzyme is oriented towards the lumen of the cisternal space. In this orientation, it catalyzes the transfer of galactose to glycoprotein-bound acetylglucosamine and, in the presence of alpha-lactalbumin, to glucose, as shown in the Golgi complex of mammary gland epithelial cells. The enzymatic properties of GT are well known. The metabolism of GT has been extensively studied in HeLa and human hepatoma cells. The enzyme is synthesized in the rough endoplasmic reticulum (RER) and provided with one N-linked oligosaccharide and palmitate residues. In the Golgi complex, terminal sugars are attached to the N-linked oligosaccharide and extensive O-glycosylation takes place. The half-life of the enzyme is about 20 hr, after which a soluble form appears in the culture medium. Release of GT into the medium is observed in all cell lines studied. This phenomenon is in accordance with the presence of soluble GT in body fluids such as serum, ascites, milk, and saliva. In patients suffering from ovarian and breast cancer, increased levels of GT enzyme activity have been reported. Whether extracellular GT is of biological significance is still a point of discussion.
TL;DR: This chapter describes protocols for purification of the enzyme from bovine milk and human postheparin plasma and presents a versatile assay procedure that can be adopted for tissue extracts, column effluents, cell cultures, postheParin plasma samples, and adipose tissue biopsy specimens.
Abstract: Publisher Summary This chapter describes protocols for purification of the enzyme from bovine milk and human postheparin plasma. It also presents a versatile assay procedure that can be adopted for tissue extracts, column effluents, cell cultures, postheparin plasma samples, and adipose tissue biopsy specimens. The enzyme has attracted considerable clinical interest as various causes of hypertriglyceridemia have been associated with impairment of its function. Lipoprotein lipase (LPL) in adipose tissue has been implicated as a major regulator of fat cell size. After the introduction of affinity chromatography on heparinagarose, several purification procedures adopting that technique have appeared and made it possible to purify the enzyme to homogeneity and to investigate its chemical and functional properties in more detail. LPL requires the presence of a specific protein cofactor, also called activator, to hydrolyze a triglyceride emulsion at an optimal rate. Maximal enzyme activity requires a sufficient concentration of triglyceride substrate and an optimal concentration of activator. The assay described herein is designed by that principle using a serum activator and radioactive triolein emulsified with lecithin.