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The Journal of Biological Chemistry Dec 1999Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was...
Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.
Topics: Animals; Enzyme Inhibitors; Glycine N-Methyltransferase; Hydrogen-Ion Concentration; Kinetics; Liver; Methyltransferases; Pancreas; Protein Binding; Pteroylpolyglutamic Acids; Rats; Tetrahydrofolates
PubMed: 10608809
DOI: 10.1074/jbc.274.53.37559 -
Cell Transplantation 2020For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be...
For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.
Topics: Animals; Collagenases; Islets of Langerhans; Islets of Langerhans Transplantation; Pancreas; Swine; Tissue Survival
PubMed: 33267618
DOI: 10.1177/0963689720977835 -
Proceedings of the National Academy of... Apr 2018Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic...
Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at "canonical" position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1' substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.
Topics: Carboxypeptidases; Catalytic Domain; Crystallography, X-Ray; Humans; Pancreas; Protease Inhibitors; Substrate Specificity
PubMed: 29636417
DOI: 10.1073/pnas.1803685115 -
Molecules (Basel, Switzerland) Oct 2022Enzymes are difficult to recycle, which limits their large-scale industrial applications. In this work, an ionic liquid-modified magnetic metal-organic framework...
Enzymes are difficult to recycle, which limits their large-scale industrial applications. In this work, an ionic liquid-modified magnetic metal-organic framework composite, IL-FeO@UiO-66-NH, was prepared and used as a support for enzyme immobilization. The properties of the support were characterized with X-ray powder diffraction (XRD), Fourier-transform infrared (FTIR) spectra, transmission electron microscopy (TEM), scanning electronic microscopy (SEM), and so on. The catalytic performance of the immobilized enzyme was also investigated in the hydrolysis reaction of glyceryl triacetate. Compared with soluble porcine pancreatic lipase (PPL), immobilized lipase (PPL-IL-FeO@UiO-66-NH) had greater catalytic activity under reaction conditions. It also showed better thermal stability and anti-denaturant properties. The specific activity of PPL-IL-FeO@UiO-66-NH was 2.3 times higher than that of soluble PPL. After 10 repeated catalytic cycles, the residual activity of PPL-IL-FeO@UiO-66-NH reached 74.4%, which was higher than that of PPL-FeO@UiO-66-NH (62.3%). In addition, kinetic parameter tests revealed that PPL-IL-FeO@UiO-66-NH had a stronger affinity to the substrate and, thus, exhibited higher catalytic efficiency. The results demonstrated that FeO@UiO-66-NH modified by ionic liquids has great potential for immobilized enzymes.
Topics: Swine; Animals; Lipase; Ionic Liquids; Enzymes, Immobilized; Metal-Organic Frameworks; Pancreas; Magnetic Phenomena; Enzyme Stability
PubMed: 36296392
DOI: 10.3390/molecules27206800 -
Poultry Science Aug 2015The purpose of the study was to examine the effect of 2 prebiotics and 2 synbiotics on the digestive potency of pancreas in 1-, 3-, 7-, 14-, 21-, and 34-day-old...
The purpose of the study was to examine the effect of 2 prebiotics and 2 synbiotics on the digestive potency of pancreas in 1-, 3-, 7-, 14-, 21-, and 34-day-old cockerels. Prebiotics (inulin and Bi²tos) and synbiotics (inulin + Lactococcus lactis subsp. lactis and Bi²tos + Lactococcus lactis subsp. cremoris) were injected in ovo into the air cell on the 12th d embryonic development. Their application increased the activity of amylase, lipase, and trypsin in the pancreas. The most pronounced changes were observed at the end of the investigated rearing period (d 34). The strongest stimulative effects on amylase were shown by both synbiotics, on lipase synbiotic Bi²tos + Lactococcus lactis subsp. cremoris, and on trypsin all the used prebiotics and synbiotics. Simultaneously, neither the absolute nor the relative mass of the pancreas in comparison to control group were changed. Also, the injected in ovo compounds did not cause a deterioration in the posthatching condition of the chicken liver, as determined by measurement of the activity of marker enzymes in the blood (alanine aminotransferase and aspartate aminotransferase). Treatment with the prebiotics and synbiotics did not change the feed conversion ratio but Bi²tos (galacto-oligosaccharide) and inulin (fructan) + Lactococcus lactis subsp. lactis significantly increased final BW.
Topics: Amylases; Animals; Chick Embryo; Chickens; Male; Pancreas; Prebiotics; Synbiotics
PubMed: 26112038
DOI: 10.3382/ps/pev162 -
Lipids in Health and Disease Dec 2011diabetes is a serious health problem and a source of risk for numerous severe complications such as obesity and hypertension. Treatment of diabetes and its related...
BACKGROUND
diabetes is a serious health problem and a source of risk for numerous severe complications such as obesity and hypertension. Treatment of diabetes and its related diseases can be achieved by inhibiting key digestives enzymes-related to starch digestion secreted by pancreas.
METHODS
The formulation omega-3 with fenugreek terpenenes was administrated to surviving diabetic rats. The inhibitory effects of this oil on rat pancreas α-amylase and maltase and plasma angiotensin-converting enzyme (ACE) were determined.
RESULTS
the findings revealed that administration of formulation omega-3 with fenugreek terpenenes (Om3/terp) considerably inhibited key enzymes-related to diabetes such as α-amylase activity by 46 and 52% and maltase activity by 37 and 35% respectively in pancreas and plasma. Moreover, the findings revealed that this supplement helped protect the β-Cells of the rats from death and damage. Interestingly, the formulation Om3/terp modulated key enzyme related to hypertension such as ACE by 37% in plasma and kidney. Moreover administration of fenugreek essential oil to surviving diabetic rats improved starch and glucose oral tolerance additively. Furthermore, the Om3/terp also decreased significantly the glucose, triglyceride (TG) and total-cholesterol (TC) and LDL-cholesterol (LDL-C) rates in the plasma and liver of diabetic rats and increased the HDL-Cholesterol (HDL-Ch) level, which helped maintain the homeostasis of blood lipid.
CONCLUSION
overall, the findings of the current study indicate that this formulation Om3/terp exhibit attractive properties and can, therefore, be considered for future application in the development of anti-diabetic, anti-hypertensive and hypolipidemic foods.
Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Drug Combinations; Fatty Acids, Omega-3; Glycoside Hydrolase Inhibitors; Hypertension; Hypoglycemic Agents; Lipids; Male; Pancreas; Peptidyl-Dipeptidase A; Plant Oils; Rats; Rats, Wistar; Seeds; Terpenes; Trigonella; alpha-Amylases; alpha-Glucosidases
PubMed: 22142357
DOI: 10.1186/1476-511X-10-226 -
The Journal of Biological Chemistry Jan 1979The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of...
The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.
Topics: Animals; Binding Sites; Carboxypeptidases; Cattle; Cobalt; Esters; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mathematics; Pancreas; Temperature; Zinc
PubMed: 33168
DOI: No ID Found -
Proceedings of the National Academy of... May 2009The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates...
The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe (gir) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.
Topics: Animals; Cell Lineage; Cell Transplantation; Cytochrome P-450 Enzyme System; Endoderm; Gene Expression Regulation, Developmental; In Situ Hybridization; Models, Biological; Mutation; Oligonucleotide Array Sequence Analysis; Pancreas; Retinoic Acid 4-Hydroxylase; Signal Transduction; Zebrafish; Zebrafish Proteins
PubMed: 19416885
DOI: 10.1073/pnas.0813108106 -
Journal of Biochemistry Nov 19791. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the...
1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 AND 5.3) WITH 0.5 M acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 M 3,3-dimethyl glutarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of the pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fmately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases. The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.07 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.
Topics: Amylases; Animals; Carboxypeptidases; Chromatography, Ion Exchange; Deoxyribonucleases; Kallikreins; Lipase; Pancreas; Solvents; Swine; Trypsin; alpha-Amylases
PubMed: 316432
DOI: 10.1093/oxfordjournals.jbchem.a132671 -
Journal of Lipid Research May 1979A systematic study has shown the importance of the different factors which are concerned with the action of lipase on a substrate (1,3-didecanoylglycerol). These consist...
A systematic study has shown the importance of the different factors which are concerned with the action of lipase on a substrate (1,3-didecanoylglycerol). These consist of a) the process of adsorption of lipase to the surface, b) the necessity of limited stirring to reach equilibrium, and c) the persistence during the reaction process of the enzyme molecules adsorbed on the monolayer. On the basis of this preliminary investigation, a technique was established to analyze the mechanism of lipase action with defined quantities of enzyme and lipid segregated in the monolayer. Thus, the process of the reaction itself is separated from the adsorption process, and it is demonstrated that the quantity of substrate hydrolyzed per minute depends only on the quantity of initially adsorbed lipase and not on the quantity of substrate or on the surface concentration of the enzyme. An appropriate new definition of the rate is consequently adopted.
Topics: Adsorption; Animals; Diglycerides; Glycerides; Kinetics; Lipase; Membranes, Artificial; Pancreas; Protein Binding; Surface Properties; Swine
PubMed: 458266
DOI: No ID Found