Purified Enzyme Preparation Research Articles

Purification and properties of an extracellular lipase fromPythium ultimum

AbstractAn extracellular triacylglycerol lipase (EC 3.1.1.3) fromPythium ultimum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL‐6B and Sephacryl S−200 chromatography. The purified enzyme preparation showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the protein was estimated at 270 kD using gel filtration on Sephacryl S−200, and 68 kD by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicating that the enzyme may be a tetramer. The optimum pH and temperature for activity of the enzyme were 8.0 and 30°C, respectively. Activity was reduced by Co2+, Fe2+, Sn2+ and Mn2+ and stimulated by Ca2+, Mg2+, Na+, K+ and surfactants such as taurocholic acid, Triton X−100,n‐octyl glucoside,n‐dodecyl‐β‐D‐maltoside, 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate(CHAPS), and 3‐[‐cholamidopropyl)dimethylammonio]‐2‐hydroxy‐1‐propanesulfonate. The apparent maximum specific activity was 42 μmole/min/mg in the absence of CHAPS and 77 μmole/min/mg in its presence. The reaction rate was progressively higher with increasing number of double bonds in the substrate, and the enzyme showed a preference for triacylglycerols containing fatty acids having thecis double bond configuration.

Isolation and characterization of an endopolygalacturonase from Phanerochaete chrysosporium

An extracellular endopolygalacturonase from extracts of solid-state cultures of Phanerochaete chrysosporium was purified to homogeneity as judged by electrophoresis under denaturing conditions. The purified enzyme preparation is a single subunit glycoprotein with an M r value of 41 700 and is comprised of three isoenzymes with pI values of 4.26, 4.46 and 4.64, respectively. It is active against polygalacturonic acid, oligogalacturonates, and to a lesser extent against pectins. Activity against the latter substrates decreases with increasing degree of methyl esterification. The enzyme readily catalyzes the hydrolysis of oligomers from tetramers upwards but acts only slowly against trimers and not at all against dimers. Examination of the pattern of products released from various substrates including oligomers suggested that the active site is comprised of six subsites. The K m for polygalacturonate is 2.5 mg ml −1, the k cat is 440 s −1, the Hill coefficient is 1.04 and the E a value is 33.4 kJ mol −1. The temperature and pH optima under assay conditions are 66°C and 4.7, respectively. The half-life of the enzyme at pH 5.0, 60°C is 14.5 min.

Physicochemical and catalytic properties of three endopolygalacturonases from Penicillium pinophilum

Three extracellular endopolygalacturonases from extracts of solid-state cultures of Penicillium pinophilum, strain ii, were purified to homogeneity as judged by electrophoresis under denaturing conditions. The purified enzyme preparations are single subunit glycoproteins with pI and M r values as follows: PG I (3.67–4.01; 43 100), PG II (4.0; 43 100), PG III (5.2–7.3; 41 600). They are all active against polygalacturonic acid, oligogalacturonates, and to a lesser extent against pectins. Activity against the latter substrates decreases with increasing degree of methyl esterification. PG I and PG III readily catalyze the hydrolysis of oligogalacturonates with DP > 4, whereas PG II acts efficiently only on those with DP > 5. The K m values (mM) for polygalacturonate are PG I (20.7), PG II (16.9), PG III (8.3); the k cat values (s −1) are PG I (91), PG II (310), PG III (4130) and in each case the Hill coefficient is unity. Temperature and pH optima under assay conditions ranged from 57 to 61°C and from 4.5 to 5.0, respectively. The E a values (kJ mol −1) were PG I (34.3), PG II (37.2) and PG III (25.7). Half-life values (min) at pH 5.0, 55°C were PG I (10.6), PG II (16.5) and PG III (9.5).

Purification and properties of the ?-3/4-l-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line

A soluble alpha-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700,000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminal N-acetylglucosamine residue in the Type 1 (Gal beta 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal beta 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any alpha-3-fucosyltransferase species acting solely on N-acetylglucosamine residues in Type 2 chains separated from the bulk of the alpha-3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal beta-galactosyl residue and 2'-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, and M(r), closely resembled those of the Lewis-blood-group gene associated alpha-3/4-fucosyltransferase isolated from human milk.

Role of cytosolic tyrosine kinase in mediating insulin-like actions of vanadate in rat adipocytes.

In previous studies we have identified a cytosolic protein tyrosine kinase (CytPTK) in rat adipocytes that is largely activated in vanadate-pretreated cells (Shisheva, A., and Shechter, Y. (1992) FEBS Lett. 300, 93-96). We report here that staurosporine and its analog K-252a are highly potent (ID50 = 3 and 100 nM, respectively) in inhibiting CytPTK activity of crude cell extract or partially purified enzyme preparations. Staurosporine and K-252a were less effective by more than 2 and 1 orders of magnitude, respectively, in inhibiting insulin receptor-catalyzed PolyGlu4Tyr phosphorylation in cell-free experiments. Preincubation of rat adipocytes with either staurosporine or K-252a selectively blocked the action of vanadate in activating glucose incorporation into lipids and its oxidation. Thus, staurosporine inhibited vanadate-stimulated lipogenesis and glucose oxidation (via glycolysis and the pentose phosphate pathway) in a concentration-dependent manner with ID50 of 75 and 300 nM, respectively. Insulin-stimulated bioeffects were not inhibited at this low range of staurosporine concentration. Staurosporine had no effect on vanadate-stimulated hexose uptake or on vanadate's antilipolytic action. Using staurosporine, we probed those insulinomimetic agents which facilitate their biological activity via the insulin receptor kinase (insulin, wheat germ agglutinin, concanavalin A, and pervanadate) or via CytPTK (vanadate and to a certain degree Mn2+ and Zn2+). These results suggest that (a) vanadate facilitates its insulin-like actions on glucose utilization via the cytosolic tyrosine kinase and (b) this enzyme does not participate in vanadate effects in stimulating hexose uptake and in inhibiting lipolysis. These findings explain further vanadate's post-insulin receptor actions and raise possible application in the management of glucose metabolism in insulin-independent fashion in pathological conditions.

Purification and characterization of recombinant human beta 1-4 galactosyltransferase expressed in Saccharomyces cerevisiae.

A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell beta-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum. Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly. This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.

Evaluation on the hydrolysis of methylumbelliferyl-tetra- N-acetylchitotetraoside by various glucosidases. A comparative study

1. 1. In human plasma, an enzyme is present which hydrolyzes 4-methylumbelliferyl-tetra- N-acetylchitotetraoside. The function of this enzyme is unknown. 2. 2. We have examined whether hyaluronidase, neutral endoglucosaminidase, N- acetyl-β- d- hexosaminidase , aspartylglucosaminidase, β- d-glucosidase, and chitobiase could hydrolyze MU-TACT. The results obtained are detailed below. 3. 3. A purified commercial preparation of hyaluronidase does not hydrolyze MU-TACT. 4. 4. Substrate specificity requirements, pH optimum and subcellular localization indicate that neutral endoglucosaminidase is distinguishable from MU-TACT hydrolase. Also commercial neutral endoglucosaminidase D and H have no affinity towards MU-TACT. 5. 5. N- Acetyl-β- d- hexosaminidase is different from MU-TACT hydrolase for the following reasons: (a) a purified enzyme preparation does not hydrolyze MU-TACT; (b) there is no correlation in the activity of the enzymes; (c) MU-TACT hydrolase is not deficient in cells of a patient with a deficiency of total N- acetyl-β- d- glucosaminidase ; and (d) the 2 enzymes have very different Chromatographic characteristics and Con A binding properties. 6. 6. Enzyme characteristics, substrate structural requirements and a lack of correlation with MU-TACT hydrolase activity suggest that aspartylglucosaminidase, β- d-glucosidase, and chitobiase are not involved in the hydrolysis of MU-TACT. 7. 7. None of the enzymes which we have considered corresponds to MU-TACT hydrolase. The exact nature and the function of the enzyme remains an enigma.

Regulation of purified type I and type II adenylylcyclases by G protein beta gamma subunits.

Type I and type II adenylylcyclases have been purified after expression in Sf9 cells, each by application of a two-step purification protocol. The specific activities of the essentially homogeneous enzymes are approximately 7 and 2 mumol.min-1.mg-1, respectively. Each purified enzyme preparation is activated by Gs alpha, but they are regulated in an opposite fashion by G protein beta gamma subunits. Purified beta gamma inhibits Gs alpha-stimulated type I adenylylcyclase directly, while beta gamma activates type II adenylylcyclase and potentiates the Gs alpha-mediated stimulation of the enzyme. This is the first demonstration of the activation of a purified effector molecule by G protein beta gamma subunits.

UDP-glucose sterol ß- d-glucosyltransferase, a plasma membrane-bound enzyme of plants: Enzymatic properties and lipid dependence

UPD-glucose sterol ß- d-glucosyltransferase (UDPG-SGTase) catalyzes the glucosylation of plant sterols. This enzyme has been shown to be membrane-bound, most of its activity being associated with plasma membrane in etiolated maize coleoptiles. After solubilization with detergents, total delipidation and purification, kinetic studies performed with a purified enzyme preparation in the presence of detergent and soybean phosphatidylcholine (PC) strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. A reduced sulfhydryl group and an arginyl residue were shown to be essential for activity. Lipid dependence studies have been performed on the delipidated enzyme in two systems: a micellar one composed of a mixture of enzyme, detergent and phospholipids and another one where the enzymatic activity was reconstituted in unilamellar lipid vesicles. In both systems it was shown that the UDPG-SGTase activity was stimulated to a large extent by negatively charged phospholipids. Enzymatic assays were performed with membrane fractions originating from plants whose sterol content was profoundly modified following treatment with a sterol biosynthesis inhibitor. Results showed that the sterol glucosylating activity was strongly inhibited in these fractions in accordance with sterol substrate specificity studies. All these results show that the UDPG-SGTase is exquisitely sensitive to its lipid environment. Physiological implications of these data are discussed in the light of the putative role of sterols in the plant cell.

7] Differentiation between central and excentric cleavage of β-carotene

A series of observations is presented in the chapter that culminate in the hypothesis that β -carotene could undergo both central and excentric cleavage. This hypothesis is partially supported by observations that the apocarotenals are effectively converted to retinol in rats as well as are being oxidized to their corresponding carotenoic acids. In addition, these apocarotenals are identified in the intestine of chickens after the administration of β -carotene and could serve as a substrate for a partially purified enzyme preparation from both rabbit and guinea pig intestine. Olson has stated that to remain viable as an alternative pathway of vitamin A formation from carotenoids in mammals, the excentric cleavage hypothesis clearly requires unambiguous direct supporting evidence. Three recent publications offer this direct supporting evidence, and the methods that they used are described in the chapter. Apart from the intrinsic interest in the fact that β -carotene can be enzymatically cleaved at several locations along the polyene chain, there is now a strong possibility that excentric cleavage may play an important role in the formation of retinoic acid. It has been shown that retinoic acid is an important metabolite of β -carotene in mammalian tissues, including the small intestine, as well as liver, kidney, lung, and testes.

Purification and characterization of a rat hepatic acetyltransferase that can metabolize aromatic amine derivatives.

Rat liver cytosol is capable of N-acetylation of arylamines, O-acetylation of arylhydroxylamines and N,O-acyltransfer of arylhydroxamic acids. The objective of this study was to characterize the enzyme(s) responsible for these reactions. A partially purified acetyltransferase preparation from rat liver cytosol was used to produce five mouse monoclonal IgG1S that bound to acetyltransferase on Western blots and affected one or more of the acetylation reactions. Two immunoaffinity columns were prepared by covalently cross-linking monoclonal antibodies to protein A-Sepharose. The first column permitted recovery of a single, immunoreactive 32 kDa protein that was capable of catalyzing all three reactions, while the second removed all three acetylation activities from a partially purified enzyme preparation and yielded a single, immunoreactive 32 kDa protein on elution. The harsh conditions necessary for elution from the latter column precluded recovery of an active enzyme. Although Western blots from SDS-PAGE at all stages of purification showed a single 32 kDa protein, purification was associated with the production of multiple, immunochemically reactive peptides with higher pIs. Direct enzymatic assays of these immunochemically reactive components after isoelectric focusing on polyacrylamide gels demonstrated that a single 32 kDa, pI 4.5 protein is capable of all three cytosolic acetylation activities. A second 32 kDa protein, pI 4.8, was able to carry out N-acetylation but not N,O-acetyltransfer. Immunoreactive components with pIs > 4.8 that were formed during purification were catalytically inactive. However, isoelectric focusing in solution of cytosolic preparations that had been subjected only to gel filtration gave a single 32 kDa immunoreactive peptide that was capable of all three acetylation reactions. Buffer concentration differentially affected the enzymatic activities of the enzyme, i.e. as a pH 7.4 buffer was decreased from 50 mM sodium pyrophosphate to 2 mM, the ability to N-acetylate arylamines was lowered while the abilities for O-acetylation and N,O-acetyltransfer were unaffected. It has been shown that a single 32 kDa protein carries out all of the acetylation reactions in rat liver cytosol. Although it cannot be ruled out that other similarly sized and closely related enzymes that share antigenic sites are also capable of these acetylation reactions, these studies suggest that instabilities of the major peptide responsible for these activities, as reflected in changes in isoelectric point, may be responsible for changes in the enzymatic potentials of this peptide.

BIOSYNTHESIS OF THE BLOOD GROUP P ANTIGEN-LIKE GalNAc^|^beta;1^|^rarr;3Gal^|^beta;1^|^rarr;4GlcNAc/Glc STRUCTURE: KINETIC EVIDENCE FOR THE RESPONSIBILITY OF N-ACETYLGLUCOSAMINYLTRANSFERASE

Previously we reported the occurrence of UDP-GalNAc:Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase activity in human plasma. Here, the donor substrate specificity of the enzyme partially purified from blood group O plasma was investigated by means of competition experiments with analogs of donor. The enzyme activity was found to be inhibited most strongly by UDP-GlcNAc among the nucleotide sugars tested, and UDP was the best inhibitor among test nucleotides. UDP-GlcNAc was much more inhibitory than UDP, whereas UDP-GalNAc was much less inhibitory than UDP. These results show that the donor-binding site of enzyme has a high affinity for UDP-GlcNAc, but not UDP-GalNAc, suggesting that the enzyme essentially functions in the transfer of N-acetylglucosamine. Indeed, UDP-GlcNAc:Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylglucosaminyltransferase activity is known to occur in human plasma. The ratio of N-acetylgalactosamine to N-acetylglucosamine transferred to lactose with O or B plasma was 1:20 when assayed separately at 1 microM of donor, but increased to 1.2:1 at 5 mM. The nearly identical ratio was obtained with the partially purified enzyme preparation.

2-Hydroxychromene-2-carboxylate isomerase from bacteria that degrade naphthalenesulfonates

2-Hydroxychromene-2-carboxylate isomerase activity was found in cell-free systems from bacteria that degrade naphthalenesulfonates. The enzyme fromPseudomonas testosteroni A3 was activated by incubation with glutathione, dithiothreitol or mercaptoethanol. The highest enzyme activity was found after preincubation of the enzyme with glutathione at alkaline pH-values. A highly purified enzyme preparation converted besides 2-hydroxychromene-2-carboxylate also 2-hydroxybenzo[g]chromene-2-carboxylate (the 2-hydroxychromene-2-carboxylate formed from 1,2-dihydroxyanthracen). The addition of various metal ions or EDTA did not significantly change the catalytic activity of the enzyme. A possible reaction mechanism is proposed.

Purification and characterization of dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures

Upon treatment of Papaveracea cells with fungal elicitors, the biosynthesis of benzo[ c]phenanthridine alkaloids is induced. Dihydrobenzophenanthridine oxidase, which catalyzes a later step in the biogenesis of these alkaloids, is one of the enzymes whose activity is elevated in the process. Here we report the 211-fold purification of the oxidase from elicited Sanguinaria canadensis by a combination of ammonium sulfate fractionation, DEAE-Sephadex, CM-Sephadex, Sephadex G-200, and either phenyl Superose or gel filtration chromatography. The purified enzyme utilized molecular oxygen to oxidize dihydrosanguinarine to sanguinarine with concomitant formation of hydrogen peroxide. A pH optimum of 7.0, V max of 27 nkat/mg protein, and apparent K m of 6.0 μ m for dihydrosanguinarine were determined. Dihydrochelerythrine was also found to be a substrate for the purified enzyme, displaying an apparent K m of 10 μ m. However, neither dihydronorsanguinarine nor the indole alkaloid ajmalicine was oxidized, indicating that the enzyme has some substrate specificity. Apparent molecular weight estimates by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis revealed that the most purified enzyme preparation obtained contained a major component of 77 kDa and two minor components between 59 and 67 kDa that can be associated with oxidase activity. Purified enzyme preparations possessed activity that was inhibited by sodium diethyldithiocarbamate, sodium azide, potassium cyanide, 1,4- dl-dithiothreitol, and mercaptoethanol.

Lysosomal β-hexosaminidase is highly resistant towards proteolytic degradation in vitro

1. 1. A partially purified enzyme preparation of β-hexosaminidase from human fibroblasts was treated with proteases and the effect on its molecular weight and enzymatic activity was studied. 2. 2. Both the forms A and B of the enzyme appeared to be resistant to a protease treatment that degraded the majority of the contaminating proteins to a large extent. 3. 3. The same result was obtained with enzyme preparations from cells treated with tunicamycin. 4. 4. Also the molecular weights of the individual polypeptide chains of the enzyme were not decreased, as was shown by SDS-PAGE, followed by immuno-blotting.

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.

Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg.min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme displayed an absolute requirement for Fe2+ and ascorbate. The stoichiometry of the enzymatic reaction was determined: ACC + ascorbate + O2-->C2H4 + HCN + CO2 + dehydroascorbate + 2 H2O. A polyclonal antibody was raised against a synthetic tridecapeptide (PDLEEEYRKTMKE) whose sequence was deduced from the apple pAE12 cDNA [Dong, J. G., Olson, D., Silverstone, A. & Yang, S. F. (1992) Plant Physiol. 98, 1530-1531], which is homologous to tomato cDNAs encoding ACC oxidase. On a Western blot, this antibody specifically recognized the purified ACC oxidase protein. The amino acid composition of the purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KEFAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.

Enzyme-catalysed synthesis of optically active aliphatic cyanohydrins

The enantioselective preparation of aliphatic ( R)-cyanohydrins was studied using powdered almond meal, a rich source of mandelonitrile lyase, as a catalyst in organic solvent. The feasibility of powdered almond meal as a catalyst was compared to that of a purified enzyme preparation.

Interest of Using a Purified, Stable and Commutable Preparation of Human Pancreatic Lipase in Indirect Assays

Abstract Human lipase has been purified from pancreatic juice. The procedure involved ammonium sulphate fractionation, anion exchange chromatography, preparative isoelectric focusing, gel filtration and cation exchange chromatography. Apparent molecular weight and pI were found to be 48 kDa and 7.5 respectively. Purified enzyme supplemented with buffered human albumin 40 g/1 and sodium azide 1 g/1 was stable in solution at 4°C for at least 200 days. The effects of bile salts, colipase and ionic strength on enzyme activity were found to be very similar regardless of the source of the enzyme (pancreatic juice or plasma of patients suffering from acute pancreatitis). Studies of commutability indicated that the purified enzyme preparation described may provide a suitable calibrator for lipase indirect assays.

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk.—III. Seasonal variation of enzymatic activity

1. 1. Fructose-1,6-bisphosphatase (FBPase-1) from the mantle and the posterior adductor muscle (PAM) of Mytilus galloprovincialis Lmk. was investigated over almost 2 years for changes in specific activity and the apparent Michaelis constant ( K m) for Fru-1,6-P 2 (purified enzyme preparations). 2. 2. The specific activity of the mantle changed, peaking in a period between August and November. 3. 3. The K m values for the substrate changed seasonally being low during the first part of the year (January–May) and high in the period between October and November. 4. 4. The changes in mantle FBPase-1 activity were discussed in relation to the effect of modulators and the possible seasonally covalent modification of the enzyme. 5. 5. No statistical changes were detected in posterior adductor muscle FBPase-1, which is in keeping with the glycolytic role suggested for the tissue.