scholarly journals The substrate specificity of acid α-glucosidase from rabbit muscle

1971 ◽  
Vol 124 (4) ◽  
pp. 701-711 ◽  
Author(s):  
T. N. Palmer
Keyword(s):  
Substrate Inhibition ◽  
Physiological Role ◽  
Liver Glycogen ◽  
Rabbit Muscle ◽  
Ph Optimum ◽  
Enzyme Action ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

1. Acid α-glucosidase was purified 3500-fold from rabbit muscle. 2. The enzyme was activated by cations, the degree of activation varying with the substrate. Enzyme action on glycogen was most strongly activated and activation was apparently of a non-competitive type. With rabbit liver glycogen as substrate, the relative Vmax. increased 15-fold, accompanied by an increase in Km from 8.3 to 68.6mm-chain end over the cation range 2–200mm-Na+ at pH4.5. Action on maltose was only moderately activated (1.3-fold, non-competitively) and action on maltotriose was marginally and competitively inhibited. 3. The pH optimum at 2mm-Na+ was 4.5 (maltose) and 5.1 (glycogen). Cation activation of enzyme action on glycogen was markedly pH-dependent. At 200mm-Na+, the pH optimum was 4.8 and activity was maximally stimulated in the range pH4.5–3.3. 4. Glucosidase action on maltosaccharides was associated with pronounced substrate inhibition at concentrations exceeding 5mm. Of the maltosaccharides tested, the enzyme showed a preference for p-nitrophenyl α-maltoside (Km 1.2mm) and maltotriose (Km 1.8mm). The extrapolated Km for enzyme action on maltose was 3.7mm. 5. The macromolecular polysaccharide substrate glycogen differed from linear maltosaccharide substrates in the kinetics of its interaction with the enzyme. Activity was markedly dependent on pH, cation concentration and polysaccharide structure. There was no substrate inhibition. 6. The enzyme exhibited constitutive α-1,6-glucanohydrolase activity. The Km for panose was 20mm. 7. The enzyme catalysed the total conversion of glycogen into glucose. The hydrolysis of α-1,6-linkages was apparently rate-limiting during the hydrolysis of glycogen. 8. Enzyme action on glycogen and maltose released the α-anomer of d-glucose. 9. The results are discussed in terms of the physiological role of acid α-glucosidase in lysosomal glycogen catabolism.

A new protease in hog thyroid lysosomes

1981 ◽  
Vol 98 (3) ◽  
pp. 390-395 ◽  
Author(s):  
H. Nakagawa ◽  
Y. Endo ◽  
S. Ohtaki
Keyword(s):  
Gel Filtration ◽  
Physiological Role ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Chloromethyl Ketone ◽  
Protein Substrates ◽  
Liver Lysosomes ◽  
Purification Ratio ◽  
Hydrolysis Of

Abstract. A leupeptin-sensitive new protease was partially purified from hog thyroid lysosomes. The purification procedure included solubilization by hypotonic treatment of lysosomes, and Sephacryl S-300 and Sephadex G-100 gel chromatography, and the purification ratio was 10-fold from lysosomes. The pH optimum of the protease activity was around 5.5 and its molecular weight was estimated to be 22 000 by gel filtration. 2-Mercaptoethanol activated the hydrolysis of protein substrates and its effect was most pronounced in the case of thyroglobulin as substrate. Among the inhibitors used, leupeptin, antipain, toluenesulfonyl-lysine chloromethyl ketone and, to a lesser degree, chymostatin and toluenesulfonyl-phenylalanine chloromethyl ketone effectively inhibited the hydrolysis of casein by the enzyme at pH 5.5, whereas pepstatin did not inhibit the activity significantly. The enzyme activity was also inhibited by sulfhydryl inhibitors such as iodoacetamide, p-chloromercuribenzoate, and N-ethylmaleimide. The release of iodoamino acids from thyroglobulin by the enzyme was inhibited in the same manner by the inhibitors used in the hydrolysis of casein. The physiological role of the new protease is discussed in comparison with cathepsin B and L found in liver lysosomes.

Angiotensin converting enzyme in brush-border membranes of avian small intestine

1988 ◽  
Vol 135 (1) ◽  
pp. 1-8
Author(s):  
B. R. Stevens ◽  
A. Fernandez ◽  
C. del Rio Martinez
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Brush Border ◽  
Physiological Role ◽  
Intestinal Epithelial ◽  
Converting Enzyme ◽  
Brush Border Membranes ◽  
Fluid Uptake ◽  
The Common ◽  
Kinetics Of ◽  
Hydrolysis Of

Angiotensin converting enzyme activity was identified in brush-border membranes purified from the small intestinal epithelium of the common grackle, Quiscalus quiscula. Angiotensin converting enzyme was enriched 20-fold in the membrane preparation, compared with intestinal epithelial cell scrapes, and was coenriched with the brush-border markers, alkaline phosphatase and aminopeptidase N. The kinetics of hydrolysis of N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG) gave a Vmax of 907 +/− 41 units g-1 and a Km of 55 +/− 6 mumol l-1. The avian intestinal angiotensin converting enzyme was inhibited by the antihypertensive drug, Ramipril, with a median inhibitory concentration (IC50) of 1 nmol l-1. In the light of previous studies on angiotensin converting enzyme in mammalian epithelia, these results may implicate a physiological role for angiotensin converting enzyme in regulating electrolyte and fluid uptake in bird small intestines.

THE ROLE OF ALKALINE PHOSPHATASE IN INTESTINAL ABSORPTION I. THE KINETICS OF PHOSPHATASE ACTION ON VARIOUS SUBSTRATES

1953 ◽  
Vol 31 (1) ◽  
pp. 1-7
Author(s):  
Neil B. Madsen ◽  
Jules Tuba
Keyword(s):  
Alkaline Phosphatase ◽  
Average Energy ◽  
Inorganic Phosphorus ◽  
Intestinal Alkaline Phosphatase ◽  
Egg Lecithin ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of intestinal alkaline phosphatase action on sodium β-glycerophosphate, glucose 6-phosphate, and egg lecithin have been studied and compared. The Michaelis constants indicate that the enzyme shows considerably less affinity for lecithin than for the other two substrates, and the approximate ratio of activity with lecithin, glucose 6-phosphate, and sodium β-glycerophosphate is 11 : 78.5 : 100. The energies of activation for the hydrolysis of the three substrates do not differ appreciably and the average energy of activation is 14,500 calories per gram-mole. The similarity of the energies of activation together with results from inhibition studies indicate that in all probability the same enzyme is responsible for the release of inorganic phosphorus from each of the three substrates.

scholarly journals Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

1972 ◽  
Vol 130 (2) ◽  
pp. 397-410 ◽  
Author(s):  
H. G. Britton ◽  
J. B. Clarke
Keyword(s):  
Chemical Equilibrium ◽  
Phosphoglycerate Mutase ◽  
Rabbit Muscle ◽  
Kinetic Measurements ◽  
Rate Limiting Step ◽  
Ping Pong ◽  
Sequential Mechanism ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Intramolecular Transfer

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

A physiological role of epidermal growth factor in cell kinetics of gastric epithelium

Life Sciences ◽  
1993 ◽  
Vol 52 (13) ◽  
pp. 1135-1139 ◽  
Author(s):  
Koichi Tasaki ◽  
Keisuke Nakata ◽  
Yuji Kato ◽  
Khaleque Newaz Khan ◽  
Satoru Mitsuoka ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Kinetics ◽  
Physiological Role ◽  
Gastric Epithelium ◽  
Epidermal Growth ◽  
Kinetics Of

scholarly journals The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1971 ◽  
Vol 124 (4) ◽  
pp. 713-724 ◽  
Author(s):  
T. N. Palmer
Keyword(s):  
Specific Binding ◽  
Ph Dependence ◽  
Thiol Group ◽  
Glycogen Storage Disease Type ◽  
Glycogen Storage ◽  
Specific Substrate ◽  
Rabbit Muscle ◽  
Ph Optimum ◽  
Enzyme Action ◽  
Substrate Binding Sites

1. The maltase and glucoamylase activities of acid α-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by αα-trehalose, methyl α-d-glucoside, sucrose, turanose, polyols, glucono-δ-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid α-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [14C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by α-1,4- and α-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [14C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

scholarly journals Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

1999 ◽  
Vol 181 (15) ◽  
pp. 4592-4597 ◽  
Author(s):  
Jeffrey A. Pederson ◽  
Gerald J. Mileski ◽  
Bart C. Weimer ◽  
James L. Steele
Keyword(s):  
Cell Surface ◽  
Deletion Mutant ◽  
Cell Envelope ◽  
Physiological Role ◽  
Lactobacillus Helveticus ◽  
Whole Cells ◽  
A Cell ◽  
Hydrolysis Of

ABSTRACT A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. TheprtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates thatprtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of αs1-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.

High-Affinity Zinc Potentiation of Inhibitory Postsynaptic Glycinergic Currents in the Zebrafish Hindbrain

2001 ◽  
Vol 85 (2) ◽  
pp. 912-925 ◽  
Author(s):  
Hiroshi Suwa ◽  
Louis Saint-Amant ◽  
Antoine Triller ◽  
Pierre Drapeau ◽  
Pascal Legendre
Keyword(s):  
Heavy Metals ◽  
Physiological Role ◽  
Synaptic Cleft ◽  
Dissociation Rate ◽  
Apparent Affinity ◽  
Zinc Binding Site ◽  
Zinc Concentrations ◽  
Kinetics Of ◽  
Whole Cell Recordings

Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC50, 0.5–1 μM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR response in control conditions. Using tricine- (10 mM) buffered zinc solution, we then showed that zinc increases the amplitude of outside-out responses evoked by 0.1–0.5 mM glycine with an EC50 of 15 nM. In contrast zinc had no effect on the amplitude of currents evoked by a saturating concentration (3–10 mM) of glycine. This suggests that zinc enhances GlyR apparent affinity for glycine. The study of the effects of zinc on the kinetics of the response indicates that this increase of apparent affinity is due to a decrease of the glycine dissociation rate constant. We then analyzed the effects of zinc on postsynaptic GlyRs in whole cell recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Chelation of contaminant heavy metals decreased the amplitude and the duration of the mIPSCs; inverse effects were observed by adding zinc in buffered solutions containing nanomolar free zinc concentrations. Zinc plus tricine or tricine alone did not change the coefficient of variation (≈0.85) of the mIPSC amplitude distributions. These results suggest that postsynaptic GlyRs are not saturated after the release of one vesicle.

scholarly journals Endogenous nicotinamide riboside metabolism protects against diet-induced liver damage

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Audrey Sambeat ◽  
Joanna Ratajczak ◽  
Magali Joffraud ◽  
José L. Sanchez-Garcia ◽  
Maria P. Giner ◽  
...  
Keyword(s):  
Metabolic Disorders ◽  
Physiological Role ◽  
Whole Body ◽  
Nicotinamide Riboside ◽  
Hepatic Cells ◽  
Broad Array ◽  
Rate Limiting ◽  
Fat Feeding ◽  
Deficient Mice

Abstract Supplementation with the NAD+ precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD+ synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD+ levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders.

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