scholarly journals Comparative study on glucocerebrosidase in spleens from patients with Gaucher disease

1990 ◽  
Vol 269 (1) ◽  
pp. 93-100 ◽  
Author(s):  
J M F G Aerts ◽  
W E Donker-Koopman ◽  
S Brul ◽  
S Van Weely ◽  
M C Sa Miranda ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Comparative Study ◽  
Molecular Mass ◽  
Gaucher Disease ◽  
Specific Activity ◽  
Normal Subjects ◽  
Accurate Assessment ◽  
Residual Enzyme Activity ◽  
Immunological Analysis ◽  
Immunological Method

In Gaucher disease (glucosylceramide lipidosis), deficiency of glucocerebrosidase causes pathological storage of glucosylceramide, particularly in the spleen. A comparative biochemical and immunological analysis has therefore been made of glucocerebrosidase in spleens from normal subjects (n = 4) and from Gaucher disease patients with non-neuronopathic (n = 5) and neuronopathic (n = 5) phenotypes. The spleens from all Gaucher disease patients showed markedly decreased glucocerebrosidase activity. Discrimination of different phenotypes of Gaucher disease was not possible on the basis of the level of residual enzyme activity, or by measurements, using the immunopurified enzyme, of kinetic constants, pI or molecular mass forms. A severe decrease was found in the specific activity of glucocerebrosidase purified to homogeneity from the spleen of a patient with the non-neuronopathic phenotype of Gaucher disease, as compared with that of the enzyme purified from the spleen of a normal subject. This finding was confirmed by an immunological method developed for accurate assessment of the relative enzyme activity per molecule of glucocerebrosidase protein. The method revealed that the residual enzyme in the spleens of all investigated patients with a non-neuronopathic course of Gaucher disease had a more than 7-fold decreased activity of glucocerebrosidase (measured in the presence of taurocholate) per molecule of enzyme, and that the concentration of glucocerebrosidase molecules in the spleens of these patients was near normal. Observations made with immunoblotting experiments were consistent with these findings. In contrast, in the spleens of patients with neuronopathic phenotypes of Gaucher disease, the concentration of glucocerebrosidase molecules was severely decreased.

A Comparative Study of the Distribution of Soluble and Particulate Glycyl-l-Leucine Hydrolase in the Small Intestine

1974 ◽  
Vol 46 (4) ◽  
pp. 501-510
Author(s):  
Manjusri Das ◽  
A. N. Radhakrishnan
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Comparative Study ◽  
Specific Activity ◽  
Maximal Activity ◽  
Total Activity ◽  
Soluble Enzyme ◽  
Km Value ◽  
Specific Activities ◽  
Very High

1. A comparative study has been made of glycyl-l-leucine hydrolase activity in the soluble and particulate fractions of intestinal mucosa from monkey, guinea-pig, rabbit and rat. The specific activity of the soluble enzyme is very high in monkey and guinea-pig, and lower in rabbit and rat. The particulate enzymes from all the four species show low specific activities and form 1–10% of the total activity. 2. The pH optima in all cases lie in the range 7·6–7·8. The Km values of the substrate were similar for both soluble and particulate enzyme from monkey and guinea-pig, but in the rabbit and rat the Km value with the particulate enzyme was higher than with the soluble enzyme. 3. The particulate enzyme activity in all cases was the highest in the distal regions of the intestine, whereas the soluble enzyme showed maximal activity in the proximal and middle regions.

scholarly journals Analysis of glucocerebrosidase activity using N-(1-[14C]hexanoyl)-d-erythro-glucosylsphingosine demonstrates a correlation between levels of residual enzyme activity and the type of Gaucher disease

1994 ◽  
Vol 303 (2) ◽  
pp. 377-382 ◽  
Author(s):  
I Meivar-Levy ◽  
M Horowitz ◽  
A H Futerman
Keyword(s):  
Enzyme Activity ◽  
Lysosomal Storage Disease ◽  
Gaucher Disease ◽  
Storage Disease ◽  
Acyl Chain ◽  
Clinical Severity ◽  
Residual Enzyme Activity ◽  
Lysosomal Storage

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutations in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of Gaucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three main types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demonstrate that a correlation exists by using a radioactive, short-acyl chain substrate, N-(1-[14C]hexanoyl)-D-erythro-glucosylsphingosine ([14C]hexanoyl-GlcCer). This substrate rapidly transferred into biological membranes in the absence of detergent [Futerman and Pagano (1991) Biochem. J. 280, 295-302] and was hydrolyzed to N-(1-[14C]hexanoyl)-D-erythro-sphingosine ([14C]hexanoyl-Cer) both in vitro and in situ, with an acid pH optimum. A strict correlation was observed between levels of [14C]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin fibroblasts. The mean residual activity measured in vitro for 3 h incubation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 +/- 4.6 nmol of [14C]hexanoyl-Cer formed per mg protein (n = 9), and in type 2 and 3 fibroblasts (the neuronopathic forms of the disease) was 19.6 +/- 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.

Inactivation of 6-phosphofructo-2-kinase during anaerobiosis in the marine whelk Busycon canaliculatum

1991 ◽  
Vol 260 (6) ◽  
pp. R1168-R1175
Author(s):  
L. Bosca ◽  
K. B. Storey
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Busycon Canaliculatum ◽  
Native Molecular Mass ◽  
Dependent Protein

6-Phosphofructo-2-kinase (PFK-2) was analyzed in four organs of the anoxia-tolerant marine gastropod mollusk Busycon canaliculatum. Whelk PFK-2 resembled the nonhepatic enzyme from mammals with highest activity occurring in gill (22 pmol.min-1.g-1). Hepatopancreas PFK-2 was purified over 8,000-fold to a final specific activity of 11 mU/mg protein (at 20 degrees C) and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a dimer with a native molecular mass of 142 kDa and a subunit molecular mass of 67 kDa. The purified enzyme showed negligible fructose-2,6-bisphosphatase (FBPase-2) activity, although the activity ratio of PFK-2 to FBPase-2 was 0.625 in crude extracts. In response to environmental anoxia, the activity of PFK-2 dropped in all organs to 34-56% of the corresponding aerobic value (half-time was 2 h in gill), and the Michaelis constant for fructose 6-phosphate increased by 50% (to 92 microM in gill). These changes paralleled decreases in organ fructose 2,6-bisphosphate concentration and pyruvate kinase activity and contribute to the overall glycolytic rate depression induced by anoxia in this facultative anaerobe. In vitro treatment of the anoxic form of hepatopancreas PFK-2 with alkaline phosphatase increased enzyme activity, suggesting that the aerobic and anoxic enzyme forms are interconverted by reversible protein phosphorylation. However, the protein kinase involved in this process is not yet known; incubation of aerobic PFK-2 with Mg-ATP plus adenosine 3',5'-cyclic monophosphate-dependent protein kinase or protein kinase C did not alter enzyme activity.

Nε-Acetyl L-α Lysine Improves Activity and Stability of α-Amylase at Acidic Conditions: A Comparative Study with other Osmolytes

2020 ◽  
Vol 27 (6) ◽  
pp. 551-556
Author(s):  
Nidhya N. Joghee ◽  
Gurunathan Jayaraman ◽  
Masilamani Selladurai
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Thermodynamic Stability ◽  
Protective Effects ◽  
Residual Enzyme Activity ◽  
Functional Stability ◽  
Biotechnological Applications ◽  
Optimal Temperature ◽  
Acidic Conditions ◽  
Cellular Components

Background: Nε-acetyl L-α lysine is an unusual acetylated di-amino acid synthesized and accumulated by certain halophiles under osmotic stress. Osmolytes are generally known to protect proteins and other cellular components under various stress conditions. Objective: The structural and functional stability imparted by Nε-acetyl L-lysine on proteins were unknown and hence was studied and compared to other commonly known bacterial osmolytes - ectoine, proline, glycine betaine, trehalose and sucrose. Methods: Effects of osmolytes on the temperature and pH profiles, pH stability and thermodynamic stability of the model enzyme, α-amylase were analyzed. Results: At physiological pH, all the osmolytes under study increased the optimal temperature for enzyme activity and improved the thermodynamic stability of the enzyme. At acidic conditions (pH 3.0), Nε-acetyl L-α lysine and ectoine improved both the catalytic and thermodynamic stability of the enzyme; it was reflected in the increase in residual enzyme activity after incubation of the enzyme at pH 3.0 for 15 min by 60% and 63.5% and the midpoint temperature of unfolding transition by 11°C and 10°C respectively. Conclusion: Such significant protective effects on both activity and stability of α-amylase imparted by addition of Nε-acetyl L-α lysine and ectoine at acidic conditions make these osmolytes interesting candidates for biotechnological applications.

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

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