Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast

Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.     

Some properties of purified phosphoprotein phosphatases from rabbit liver.

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.     

Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcusfuriosus

Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml^–1 glycogen concentration was 52 U mg^–1 and 31 U mg^–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min^–1 µg^–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.     

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella . UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Rat adipose tissue glycogen synthase. Evidence for multiple discrete kinetic species and their interconversion.

Rat adipose tissue glycogen synthase has been kinetically characterized. The classical D form has an apparent Km for UDP-glucose of 0.7 mM and 0.4 mM in the absence and presence of glucose 6-phosphate, respectively. The apparent Ka for glucose 6-phosphate is 0.6 mM. The effect of glucose 6-phosphate on the D form is to enhance the Vmax 7-fold. The I form is also affected by glucose 6-phosphate (Ka, 0.025 mM) but the Vmax is increased only by 20%; apparent Km values for UDP-glucose are 0.4 mM and 0.045 mM in the absence and presence of glucose 6-phosphate, respectively. In addition, two new kinetically distinguishable forms have been observed. The first, designated glycogen synthase Q, arises from an Mg2+ATP-dependent deactivation of the I form. The apparent Km values of glycogen synthase Q for UDP-glucose are identical with those of the I form; however, the apparent Ka for glucose 6-phosphate (0.2 mM) is 8-fold higher than that for the I form and one-third that for the D form. Preparations from fasted or diabetic rats contain a form of glycogen synthase, designated glycogen synthase X, that has a much lower affinity for glucose 6-phosphate than the D form (apparent Ka, 3 mM); the apparent Km values for UDP-glucose are similar to those of the D form (0.7 mM and 0.3 mM in the absence and presence of glucose 6-phosphate, respectively). In preparations from fasted rats a stepwise Mg2+-dependent conversion was demonstrated of synthase X to D to Q to I; this sequential conversion was reversed on incubation with Mg2+ATP. In preparations from fed rats, synthase Q could be generated either by limited activation (from the D form) or, after conversion to the I form, by deactivation with Mg2+ATP. However, even prolonged incubation with Mg2+ATP failed to generate the D (or X) form.     

Characterization of a glycosphingolipid beta-N-acetylgalactosaminyl-transferase activity in cultured hamster (nil) cells

The activity of a glycosphingolipid N-acetylgalactosaminyltransferase (GalNAc transferase) in cultured hamster fibroblasts (NIL-8) was characterized with respect to substrate binding, acceptor specificity, pH optimum and detergent requirements. Of the glycosphingolipid acceptors tested, transferase activity was observed only with globotriaosylceramide. The apparent Km values for uridinediphosphate-N-acetylgalactosamine and globotriasylceramide were 0.14 and 0.42 mM, respectively. The enzyme required Mn2+ for maximum activity (4 mM), and Mg2+ was not able to replace Mn2+. Of the detergents tested, sodium taurodeoxycholate gave the greatest activation of the enzyme at 1 mg/ml. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.0 in 2-(N-morpholino)ethanesulfonic acid. Globotetraosylceramide and II3-alpha-N-acetylneuraminyl-lactosylceramide inhibited transferase activity with globotriaosylceramide as substrate, but lactosylceramide had no effect on the activity with this acceptor. The major product of the assay was shown to be a tetraglycosylceramide with a terminal beta-N-acetylgalactosamine moiety by co-migration with authentic globotetraosylceramide on TLC plates and by cleavage of the labeled N-acetylgalactosamine from the product by jack bean beta-hexosaminidase.     

Purification and properties of human serum dopamine-β -hydroxylase

1. Two different molecular forms of dopamine-beta-hydroxylase were isolated from human serum; a major component (Peak I enzyme) with a molecular weight of 368000 and with a higher specific activity and a minor component (Peak II enzyme) with a molecular weight of 188000 and with a lower specific activity. 2. Both forms require ascorbic acid for the activity, and are stimulated by fumarate. Addition of N-ethylmaleimide or copper also increased the activity. The optimal pH of both forms in the presence of 20mM tyramine as substrate is 5.0. 3. Km values toward tyramine of Peak I enzyme and Peak II enzyme were 1.67 mM and 14.2 mM respectively. 4. Both Peak I enzyme and Peak II enzyme are glycoprotein.     

The hysteretic properties of glycogen synthase I.

Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent process (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 degrees C, the half-time of activation t 1/2 decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The Km for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process. The activation can be described by vt = vf + (vo - vf) e-kt where vt, vf and vo are velocities at times t, O and infinity and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple biolecular process: enzyme + glycogen in equilibrium enzyme-glycogen complex, with the dissociation constant Ks = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H+ is discussed.     

A comparison of the non-specific acid phosphomonoesterase activity in the larva of Phocanema decipiens (Nematoda) with that of the muscle of its host the codfish (Gadus morhua).

The non-specific phosphomonoesterase (enzyme I) extracted from the larva of the codworm (Phocanema decipiens) is different from the enzyme (enzyme II) from the muscle of its host, the codfish (Gadus morhua). The pH optima were 4.0 and 4.5, and the KM values for p-nitrophenyl phosphate hydrolysis were 1.8 mM and 6.5 mM for enzymes I and II respectively. The specific specific activity in units (0.01 mumol/min) per mg protein was 4.80 +/- 0.85 and 0.54 +/- 0.07 for enzymes I and II respectively. The specific activity from uninfected muscles was only 0.39 (SD +/- 0.017) units per mg of protein. Both enzymes were inhibited by NaF, HgCl2, and cysteine but were stimulated by 2-mercaptoethanol. EDTA and iodoacetamide had no effect on enzyme I but enzyme II was activated by EDTA and inhibited by iodoacetamide. Cadmium ions inhibited both the enzymes but a conspicuous feature with enzyme II was in the increase in percentage inhibition by lowering the concentration of CD2+.     

Properties of acid beta-N-acetyl-D-glucosaminidase from cock semen.

Two forms (I and II) of beta-N-acetyl-D-glucosaminidase from cock seminal plasma and one form (III) from spermatozoa were separated by chromatofocusing. The active enzyme forms I and II had pI values of 6.6 and 6.3, respectively, while form III had two subforms with pI values of 6.3 and 6.1, as determined by polyacrylamide gel electrofocusing. The molecular weights were 76,000 for forms I and III and 32,000 for form II. The optimum pH of enzyme forms I and III ranged from 3.6 to 4.0. In contrast, form II showed one distinct maximum at pH 3.7. The Km values obtained with p-nitrophenyl-beta-N-acetyl-D-glucosaminide as substrate were 0.35, 0.28, and 0.39 mM for forms I, II, and III, respectively. It is assumed that both cock spermatozoa and cock seminal plasma contain a common, enzymatically active beta-N-acetyl-D-glucosaminidase subunit with M(r) about 32,000 and pI 6.3.     

Physiological and enzymatic properties of the ram epididymal soluble form of germinal angiotensin I-converting enzyme.

The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril.     

The activity of yeast ADH I and ADH II with long-chain alcohols and diols

The activities of yeast ADH I and ADH II towards long chain alcohols and diols were studied using rather unusual conditions (1.0 M Tris pH 8.75, approximately 0.3 mg/ml enzyme and [S]相似文献   

A thermostable acid alpha-glucosidase from Tetrahymena thermophila: purification and characterization

An acid alpha-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56 degrees C and showed resistance to thermal inactivation. The acid alpha-glucosidase appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid alpha-glucosidase.     

Phosphorylation of Delta Sleep-Inducing Peptide (DSIP) by casein kinase II in vitro

A phosphorylated analogue of DSIP at Ser⁷ has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent K_m and V_max values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.     

Purification and properties of acid alpha-glucosidase (gamma-amylase) from the human liver]

Acid alpha-glucosidase from human liver was 720-fold purified by means of a specific sorption on Sephadex G-150 and a specific desorption from Sephadex by the competitive inhibitor, methyl-alpha-D-glucopyranoside. The preparation obtained was homogenous in ultracentrifuge and polyacrilamide gel electrophoresis. The enzyme possessed both maltase and glucoamylase activities and splitted maltose, amylopectin and glycogen with Km values of 7mM, 7.7 mg/ml and 5 mg/ml respectively. Methyl-alpha-D-glucopyranoside competitively inhibited the enzymatic hydrolysis of polysaccharides (Ki=6.95 mM) and did not affect the maltose degradation. The sedimentation coefficient of the purified enzyme preparation was 5.4 S; in 5 M guanidine. HCl the coefficient decreased to 2.2 S, which testified to the fact that the enzyme molecule consisted of subunits.     

A Thermostable Acid α-Glucosidase from Tetrahymena thermophila: Purification and Characterization

An acid α-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56° C and showed resistance to thermal inactivation. The acid α-glucosidase appears to have α-1,6-glucosidase as well as α-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-α-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid α-glucosidase.     

Purification and characterization of rat plasma kallikrein

Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.     

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg(-1) and 326 U mg(-1) respectively. The optimum pH range was from 7-8.5 at 37 degrees C and the optimum temperature was 50 degrees C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608 +/- 0.07 mM and 249 sec(-1) respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68 +/- 12.55 nM and 6.763 +/- 0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17beta-estradiol, progesterone, and prostaglandins E2 and F2alpha was determined. The data showed a modulation of 17beta-estradiol, progesterone and prostaglandin E2 production by ovary ACE.