scholarly journals The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N

1985 ◽  
Vol 231 (2) ◽  
pp. 445-449 ◽  
Author(s):  
R Matsas ◽  
S L Stephenson ◽  
J Hryszko ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Phase Separation ◽  
Membrane Protein ◽  
Total Activity ◽  
Aminopeptidase N ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Rich Phase ◽  
Triton X

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 × 10(−7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 × 10(−6) M) than for the other two forms (IC50 = 1.6 × 10(−5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 × 10(−5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 × 10(−4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

scholarly journals Purification and properties of a neurotensin-degrading endopeptidase from pig brain

1991 ◽  
Vol 276 (3) ◽  
pp. 583-591 ◽  
Author(s):  
P E Millican ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Membrane Protein ◽  
Hydrophobic Interactions ◽  
Total Activity ◽  
Integral Membrane Protein ◽  
Major Band ◽  
Pig Brain ◽  
Purification And Properties ◽  
Endopeptidase Activity ◽  
Triton X ◽  
Endopeptidase 24.15

Neurotensin (NT) endopeptidase (EC 3.4.24.16) has been purified about 800-fold from pig brain by four sequential chromatographic steps depending on ion-exchange and hydrophobic interactions. Two types of preparation were studied: one from a Triton X-100-solubilized membrane fraction, and the other from the soluble fraction containing 90% or more of the total activity in the homogenate. NT endopeptidase activity was monitored by high-precision liquid chromatography of the two peptide products, characterized as NT-(1-10) and NT-(1-8), resulting from cleavage of the Pro10-Tyr11 and Arg8-Arg9 bonds respectively. As purification proceeded, from both membranes and cytosol, the yield of the two products achieved a constant ratio of 5:1 and this ratio was reproduced in repeated purifications. However, a distinct peptidase which hydrolysed exclusively at the Arg8-Arg9 bond was partially resolved from NT endopeptidase by chromatography on hydroxyapatite, and this activity was further purified and assigned to endopeptidase-24.15 (EC 3.4.24.15). SDS/PAGE of both preparations of neurotensin endopeptidase revealed a major band of apparent Mr 75000, and treatment of the membrane-associated form with N-Glycanase gave no evidence that the enzyme was a glycoprotein. The membrane-associated and cytosol forms of NT endopeptidase activities, monitored for both NT-(1-10) and NT-(1-8) products, were compared in their responses to 1,10-phenanthroline, EDTA, dithiothreitol (DTT) and some synthetic site-directed inhibitors of endopeptidase-24.15 or peptidyl dipeptidase A. The effects revealed no significant differences between the two preparations, nor did the reagents discriminate between the activities generating the two NT fragments. The partially purified form of endopeptidase-24.15 was also included in this comparison: while some responses were similar, this peptidase was distinguishable in its activation by DTT and its relative resistance to inhibition by EDTA. Both forms of NT endopeptidase were found to hydrolyse other substrates, including Boc-Phe-Ala-Ala-Phe-4-aminobenzoate, bradykinin and substance P (these at faster rates than neurotensin), as well as dynorphin A-(1-8) and luliberin. The bonds hydrolysed in these neuropeptides, as well as in angiotensins I and II and alpha-neoendorphin, were defined. These studies confirm that NT endopeptidase is distinct from endopeptidase-24.15. They further show that the former is a soluble enzyme, not an integral membrane protein, that it is not peptide-specific and that it might be more appropriately named. enzyme, not an integral membrane protein, that it is not peptide-specific and

scholarly journals Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Application to subcellular fractions of the adrenal medulla

1986 ◽  
Vol 233 (2) ◽  
pp. 525-533 ◽  
Author(s):  
J G Pryde ◽  
J H Phillips
Keyword(s):  
Endoplasmic Reticulum ◽  
Phase Separation ◽  
Membrane Proteins ◽  
Low Temperature ◽  
Membrane Glycoproteins ◽  
Chromaffin Granule ◽  
Rich Phase ◽  
Granule Membrane ◽  
Separation Of Proteins ◽  
Triton X

After solubilization with the detergent Triton X-114, membrane proteins may be separated into three groups: if the membrane is sufficiently lipid-rich, one family of hydrophobic constituents separates spontaneously at low temperature; warming at 30 degrees C leads to separation of a detergent-rich phase and an aqueous phase. Using the chromaffin-granule membrane as a model, we found that many intrinsic membrane glycoproteins are found in the latter phase, probably maintained in solution by adherent detergent. They precipitate, however, when this is removed by dialysis, leaving in solution those truly hydrophilic proteins that were originally adhering to the membranes. We have used this method with mitochondria, and with Golgi- and rough-endoplasmic-reticulum-enriched microsomal fractions: it has proved to be a rapid and convenient method for effecting a partial separation of proteins from a variety of different membranes.

scholarly journals Aminopeptidase activity is related to the amino acids composition of the food in passerine birds

2017 ◽  
Author(s):  
Cintia Garro ◽  
Antonio Brun ◽  
Enrique Caviedes-Vidal
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Feeding Habits ◽  
Adaptive Modulation ◽  
Aminopeptidase N ◽  
Enzyme Hydrolysis ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Passerine Birds ◽  
Natural Diet

Background. Passerine birds exploit different kinds of feeding habits and they have to face seasonal changes in food availability. Therefore, the composition of the principal nutrient in their food differs from the usual. In consequence the digestive function – enzyme hydrolysis and absorption – have to adapt to these nutrients. These changes in digestive physiology could respond to the adaptive modulation hypothesis which postulated that the activities of digestive enzymes should match the levels of their substrates in their diet so energy is not wasted on enzymes that are no need. Thus, we decide to measure intestinal enzymes activities of two species of passerine birds that differ in natural diet. Overall we hypothesized that species with different feeding habits present enzyme activity according to the mainly component of the diet (e.g., carbohydrates, proteins). Our prediction is that the individuals will present enzyme activity proportionally to the primary components of the diets. Methods. We select for study: red ovenbirds (Furnarius rufus), which are strict insectivores and zebra finches (Taeniopygia guttata), which are specialist granivores. We complete the analysis with publish data for house sparrows (Passser domesticus) feed on high starch from the literature. To examine intestinal enzyme activities, we measured the activity of two disaccharidases (sucrase-isomaltase and maltase-glucoamilase) and one dipeptidase (aminopeptidase-N). Results. The average intestinal activity of sucrase shows that the omnivorous P. domesticus presents almost 4 times more activity than the granivorous T. guttata and more than 11 times than the insectivorous F. rufus. This difference is also reflected in the total sucrase hydrolytic capacity where P. domesticus has roughly 10 times more than the other two birds. Surprisingly in F. rufus we found maltase and aminopeptidase activity while sucrase activity was close to zero. In the case of the average activity of maltase for the omnivorous P. domesticus is approximately 40 % more than the granivorous T. guttata and more than 5 times than the insectivorous F. rufus. Although the total maltase hydrolytic capacity of P. domesticus is 5 times more than T. guttata and F. rufus. The average of aminopeptidase-N activity for F. rufus and T. guttata almost doubled the P. domesticus ones. Also F. rufus roughly doubles the other two birds in total aminopeptidase hydrolytic capacity. Discussion. This study has shown that exist a relationship between the levels of amino acids in the diet and the total aminopeptidase capacity, but in the case of carbohydrates this relationship is not evident.

scholarly journals Recovery of Active Polyphenol Oxidase and Peroxidase from Plant Tissues with High Phenolics and Chlorophylls

2018 ◽  
Author(s):  
Sarana Rose Sommano
Keyword(s):  
Phase Separation ◽  
High Temperature ◽  
Enzymatic Activity ◽  
Polyphenol Oxidase ◽  
Plant Tissues ◽  
Rich Phase ◽  
Post Harvest ◽  
Membrane Bound ◽  
Triton X ◽  
High Enzymatic Activity

The present protocol described extraction of active polyphenol oxidase and peroxidase from a plant rich in phenolics and chlorophylls in the post-harvest browning syndrome of B. myrtifolia.  Initially, general optimisation using conventional enzyme extractions was performed.  However, along with membrane-bound proteins, chlorophylls and phenols were also released with Triton X (TTX).  With a view to obtaining high enzymatic activity, removal of the released chlorophylls and phenols by formation of TTX-114 micelles in the detergent rich phase after high-temperature induced phase separation was tested.

Endoplasmin is a reticuloplasmin

1988 ◽  
Vol 90 (3) ◽  
pp. 485-491
Author(s):  
G.L. Koch ◽  
D.R. Macer ◽  
F.B. Wooding
Keyword(s):  
Endoplasmic Reticulum ◽  
Phase Separation ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Immunogold Labelling ◽  
Soluble Form ◽  
Intact Molecule ◽  
Carboxy Terminal ◽  
Triton X ◽  
Separation Test

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino- and carboxy-terminal sequences of the intact molecule, showing that it has not been proteolysed. Application of the Triton X-114 phase-separation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplasmic reticulum (ER) lumen show that endoplasmin is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.

scholarly journals Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes

1988 ◽  
Vol 255 (3) ◽  
pp. 843-847 ◽  
Author(s):  
R E Isaac
Keyword(s):  
Schistocerca Gregaria ◽  
Physiological Role ◽  
Aminopeptidase Activity ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Adipokinetic Hormone ◽  
Peptide Bonds ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity ◽  
Neural Membranes

Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.

scholarly journals Aminopeptidase activity is related to the amino acids composition of the food in passerine birds

2017 ◽  
Author(s):  
Cintia Garro ◽  
Antonio Brun ◽  
Enrique Caviedes-Vidal
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Feeding Habits ◽  
Adaptive Modulation ◽  
Aminopeptidase N ◽  
Enzyme Hydrolysis ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Passerine Birds ◽  
Natural Diet

Background. Passerine birds exploit different kinds of feeding habits and they have to face seasonal changes in food availability. Therefore, the composition of the principal nutrient in their food differs from the usual. In consequence the digestive function – enzyme hydrolysis and absorption – have to adapt to these nutrients. These changes in digestive physiology could respond to the adaptive modulation hypothesis which postulated that the activities of digestive enzymes should match the levels of their substrates in their diet so energy is not wasted on enzymes that are no need. Thus, we decide to measure intestinal enzymes activities of two species of passerine birds that differ in natural diet. Overall we hypothesized that species with different feeding habits present enzyme activity according to the mainly component of the diet (e.g., carbohydrates, proteins). Our prediction is that the individuals will present enzyme activity proportionally to the primary components of the diets. Methods. We select for study: red ovenbirds (Furnarius rufus), which are strict insectivores and zebra finches (Taeniopygia guttata), which are specialist granivores. We complete the analysis with publish data for house sparrows (Passser domesticus) feed on high starch from the literature. To examine intestinal enzyme activities, we measured the activity of two disaccharidases (sucrase-isomaltase and maltase-glucoamilase) and one dipeptidase (aminopeptidase-N). Results. The average intestinal activity of sucrase shows that the omnivorous P. domesticus presents almost 4 times more activity than the granivorous T. guttata and more than 11 times than the insectivorous F. rufus. This difference is also reflected in the total sucrase hydrolytic capacity where P. domesticus has roughly 10 times more than the other two birds. Surprisingly in F. rufus we found maltase and aminopeptidase activity while sucrase activity was close to zero. In the case of the average activity of maltase for the omnivorous P. domesticus is approximately 40 % more than the granivorous T. guttata and more than 5 times than the insectivorous F. rufus. Although the total maltase hydrolytic capacity of P. domesticus is 5 times more than T. guttata and F. rufus. The average of aminopeptidase-N activity for F. rufus and T. guttata almost doubled the P. domesticus ones. Also F. rufus roughly doubles the other two birds in total aminopeptidase hydrolytic capacity. Discussion. This study has shown that exist a relationship between the levels of amino acids in the diet and the total aminopeptidase capacity, but in the case of carbohydrates this relationship is not evident.

scholarly journals Analysis of toxoplasma gondii proteins after Triton X-114 solubilization and hidropholic chromotograhy

1988 ◽  
Vol 83 (4) ◽  
pp. 513-517 ◽  
Author(s):  
Salvatore Giovanni De Simone ◽  
Helena C. B. Guedes ◽  
Izidro Bendet
Keyword(s):  
Phase Separation ◽  
Toxoplasma Gondii ◽  
Human Serum ◽  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Surface Proteins ◽  
Isolation Method ◽  
Rich Phase ◽  
Sepharose Column ◽  
Triton X

The distribution of the surface proteins of toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Twopolypeptides with 15,000 and 70,000 distributed on both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities.two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of t. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.

scholarly journals Aminopeptidase activity is related to the amino acids composition of the food in passerine birds

2017 ◽  
Author(s):  
Cintia Garro ◽  
Antonio Brun ◽  
William Karasov ◽  
Enrique Caviedes-Vidal
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Feeding Habits ◽  
Adaptive Modulation ◽  
Aminopeptidase N ◽  
Enzyme Hydrolysis ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Passerine Birds ◽  
Natural Diet

Background. Passerine birds exploit different kinds of feeding habits and they have to face seasonal changes in food availability. Therefore, the composition of the principal nutrient in their food differs from the usual. In consequence the digestive function – enzyme hydrolysis and absorption – have to adapt to these nutrients. These changes in digestive physiology could respond to the adaptive modulation hypothesis which postulated that the activities of digestive enzymes should match the levels of their substrates in their diet so energy is not wasted on enzymes that are no need. Thus, we decide to measure intestinal enzymes activities of two species of passerine birds that differ in natural diet. Overall we hypothesized that species with different feeding habits present enzyme activity according to the mainly component of the diet (e.g., carbohydrates, proteins). Our prediction is that the individuals will present enzyme activity proportionally to the primary components of the diets. Methods. We select for study: red ovenbirds (Furnarius rufus), which are strict insectivores and zebra finches (Taeniopygia guttata), which are specialist granivores. We complete the analysis with publish data for house sparrows (Passser domesticus) feed on high starch from the literature. To examine intestinal enzyme activities, we measured the activity of two disaccharidases (sucrase-isomaltase and maltase-glucoamilase) and one dipeptidase (aminopeptidase-N). Results. The average intestinal activity of sucrase shows that the omnivorous P. domesticus presents almost 4 times more activity than the granivorous T. guttata and more than 11 times than the insectivorous F. rufus. This difference is also reflected in the total sucrase hydrolytic capacity where P. domesticus has roughly 10 times more than the other two birds. Surprisingly in F. rufus we found maltase and aminopeptidase activity while sucrase activity was close to zero. In the case of the average activity of maltase for the omnivorous P. domesticus is approximately 40 % more than the granivorous T. guttata and more than 5 times than the insectivorous F. rufus. Although the total maltase hydrolytic capacity of P. domesticus is 5 times more than T. guttata and F. rufus. The average of aminopeptidase-N activity for F. rufus and T. guttata almost doubled the P. domesticus ones. Also F. rufus roughly doubles the other two birds in total aminopeptidase hydrolytic capacity. Discussion. This study has shown that exist a relationship between the levels of amino acids in the diet and the total aminopeptidase capacity, but in the case of carbohydrates this relationship is not evident.

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