scholarly journals Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay

1992 ◽  
Vol 286 (1) ◽  
pp. 223-229 ◽  
Author(s):  
E M Bailyes ◽  
J C Hutton
Keyword(s):  
Gel Filtration ◽  
Sensitive Assay ◽  
Reaction Products ◽  
Ph Optimum ◽  
Insulin Granules ◽  
Proinsulin Processing ◽  
A Chain ◽  
Triton X

A simple, rapid and sensitive assay for the type-1 endopeptidase (Arg-Arg cleaving) was developed by using an antiproinsulin monoclonal immunoadsorbent to separate reaction products from the substrate. The values obtained by this assay were identical with those obtained by an h.p.l.c.-based procedure and yielded similar values for the pH optimum (5.6) and Ca2+ activation (K0.5 = 2 mM). It was shown that the type-1 endopeptidase was readily solubilized by Triton X-114 (87 +/- 3%, n = 12) and partitioned principally into the aqueous phase at 30 degrees C (90.1 +/- 2.6%, n = 12). Activity was lost on gel filtration, but could be restored by adenosine 5′-[gamma-thio]triphosphate (K0.5 = 6 microM), 50 microM-dithiothreitol or 50 microM-Ca(2+)-trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid (CDTA), indicating that the enzyme was particularly sensitive to heavy metal ions. The Km obtained with proinsulin as substrate (13 +/- 1.7 microM) indicated that the enzyme works at close to its Vmax. in the nascent secretory granule. The Vmax. of the enzyme prepared from insulin granules (0.6% proinsulin converted/min) corresponded closely to the rate measured in vivo in rat islets. The type-1 endopeptidase also appears to be capable of binding to proinsulin in the region of the C-peptide/A-chain junction, since a peptide spanning this region was found to inhibit the 125I-proinsulin processing measured by this assay.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

scholarly journals Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

1985 ◽  
Vol 229 (3) ◽  
pp. 679-685 ◽  
Author(s):  
R L Hopfer ◽  
J A Alhadeff
Keyword(s):  
Human Brain ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Gel Filtration ◽  
Compelling Evidence ◽  
Supernatant Fluid ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Supernatant

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at −20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

scholarly journals Sustained high levels of circulating chaperoned interleukin-6 after active specific cancer immunotherapy

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1887-1895 ◽  
Author(s):  
LT May ◽  
K Patel ◽  
D Garcia ◽  
MI Ndubuisi ◽  
S Ferrone ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Phase 1 ◽  
Gel Filtration ◽  
Clinical History ◽  
Active Immunization ◽  
Biologically Active ◽  
Serum Samples

Abstract In a phase 1 study of recombinant interleukin-6 (rIL-6) in patients with advanced solid tumors (n = 15), we discovered that the endogenous IL-6 levels, in pretreatment plasma or serum samples, were distributed into two groups. One set of patients (designated “type 1”; n = 9) was characterized by low plasma IL-6 levels (48 to 1,700 pg/mL) as measured using enzyme-linked immunosorbent assays (ELISA) for IL-6. In the second set of patients (designated “type 2”; n = 6), IL-6 ELISAs showed high levels of plasma IL-6 (50 to 600 ng/mL). Neither group had detectable B9 hybridoma cell growth factor activity associated with the IL-6 in their pretreatment plasma or serum. Plasma C-reactive protein (CRP) levels were markedly elevated in type II patients suggesting that the circulating IL-6 was biologically active in vivo. In both groups of patients there was a small but significant increase in B9 activity in the plasma within three hours after rIL-6 administration (n = 5). Gel filtration profiles showed that circulating IL-6 in type 1 patients, 15 to 120 minutes after rIL-6 administration was of approximate mass 20 to 40 kD, whereas in type 2 patients, the IL-6 before and after exogenous rIL-6 administration was indistinguishable and was of an approximate mass of 200 kD. IL-6 immunoaffinity purification of the 200 kD complexes showed these to contain multiple isoforms of IL-6 (14 to 31 kD) and the soluble IL-6 receptor (sIL-6R; 50 to 55 kD). A distinguishing clinical history was that all of the type 2 patients had been actively immunized with an anti-idiotypic monoclonal antibody (MoAb) (MK2–23) 3 to 12 months before initiation of this study for advanced melanoma. An analysis of the plasma IL-6 content in other melanoma patients (n = 16) during antiidiotypic MoAb immunization indicated that marked (up to 600 ng/mL) and sustained (several months) elevations of circulating “chaperoned” IL-6 were induced by active immunization regimens.

scholarly journals Properties of rat heart adenosine kinase

1984 ◽  
Vol 221 (2) ◽  
pp. 521-528 ◽  
Author(s):  
M N Fisher ◽  
E A Newsholme
Keyword(s):  
Rat Heart ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Adenosine Kinase ◽  
Ph Optimum ◽  
Metabolizing Enzymes ◽  
Adenosine Concentration ◽  
Concentration Changes ◽  
Kinase A

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.

scholarly journals Sustained high levels of circulating chaperoned interleukin-6 after active specific cancer immunotherapy

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1887-1895 ◽  
Author(s):  
LT May ◽  
K Patel ◽  
D Garcia ◽  
MI Ndubuisi ◽  
S Ferrone ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Phase 1 ◽  
Gel Filtration ◽  
Clinical History ◽  
Active Immunization ◽  
Biologically Active ◽  
Serum Samples

In a phase 1 study of recombinant interleukin-6 (rIL-6) in patients with advanced solid tumors (n = 15), we discovered that the endogenous IL-6 levels, in pretreatment plasma or serum samples, were distributed into two groups. One set of patients (designated “type 1”; n = 9) was characterized by low plasma IL-6 levels (48 to 1,700 pg/mL) as measured using enzyme-linked immunosorbent assays (ELISA) for IL-6. In the second set of patients (designated “type 2”; n = 6), IL-6 ELISAs showed high levels of plasma IL-6 (50 to 600 ng/mL). Neither group had detectable B9 hybridoma cell growth factor activity associated with the IL-6 in their pretreatment plasma or serum. Plasma C-reactive protein (CRP) levels were markedly elevated in type II patients suggesting that the circulating IL-6 was biologically active in vivo. In both groups of patients there was a small but significant increase in B9 activity in the plasma within three hours after rIL-6 administration (n = 5). Gel filtration profiles showed that circulating IL-6 in type 1 patients, 15 to 120 minutes after rIL-6 administration was of approximate mass 20 to 40 kD, whereas in type 2 patients, the IL-6 before and after exogenous rIL-6 administration was indistinguishable and was of an approximate mass of 200 kD. IL-6 immunoaffinity purification of the 200 kD complexes showed these to contain multiple isoforms of IL-6 (14 to 31 kD) and the soluble IL-6 receptor (sIL-6R; 50 to 55 kD). A distinguishing clinical history was that all of the type 2 patients had been actively immunized with an anti-idiotypic monoclonal antibody (MoAb) (MK2–23) 3 to 12 months before initiation of this study for advanced melanoma. An analysis of the plasma IL-6 content in other melanoma patients (n = 16) during antiidiotypic MoAb immunization indicated that marked (up to 600 ng/mL) and sustained (several months) elevations of circulating “chaperoned” IL-6 were induced by active immunization regimens.

scholarly journals Membrane protein phosphotyrosine phosphatase in rabbit kidney. Proteolysis activates the enzyme and generates soluble catalytic fragments

1987 ◽  
Vol 243 (3) ◽  
pp. 747-754 ◽  
Author(s):  
S A Rotenberg ◽  
D L Brautigan
Keyword(s):  
Phosphatase Activity ◽  
Plasma Membranes ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Cell Types ◽  
Total Activity ◽  
Rabbit Kidney ◽  
Ph Optimum ◽  
Fresh Tissue

Most protein phosphotyrosine phosphatases (PPT-phosphatases) have been recovered from the cytosol of various cell types and tissues. The present study explores the properties of PPT-phosphatases in rabbit kidney membranes prepared by centrifugation at 100,000 g. More of the total activity was recovered in membranes from fresh (45%) compared with frozen-and-thawed (36%) tissue. However, extracts of fresh tissue had only 15-30% as much total PPT-phosphatase activity. Up to 3-fold activation of cytosolic and membrane PPT-phosphatases occurred during preparation, an effect most evident when fresh tissue was homogenized in buffers containing multiple proteinase inhibitors. These inhibitors apparently block some, but not all, digestion of proteins that mask PPT-phosphatase activity. Incubation of membranes prepared from fresh tissue with added trypsin, papain or thermolysin in each case caused activation of PPT-phosphatase as well as generation of a soluble catalytic fragment. The fragment also was generated by the action of endogenous proteinases during repeated centrifugation and was isolated from these supernatants by DEAE-Sepharose, Zn2+-affinity and gel-filtration chromatography. The fragment had Mr approx. 33,000, had a neutral pH optimum, was inhibited by 50% by 100 microM-vanadate, and was insensitive to the alkaline-phosphatase inhibitors EDTA and levamisole. Although the chromatographic behaviour and lability of the fragment were distinct from those of the predominant cytosolic PPT-phosphatase, some cytosolic PPT-phosphatases exhibited properties consistent with the suggestion that they are fragments derived by proteolysis of PPT-phosphatases in membranes. Localization of PPT-phosphatases in plasma membranes would facilitate reaction with receptor/kinases in vivo.

scholarly journals Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

1999 ◽  
Vol 181 (10) ◽  
pp. 3129-3135 ◽  
Author(s):  
Maria Sandkvist ◽  
Lloyd P. Hough ◽  
Mira M. Bagdasarian ◽  
Michael Bagdasarian
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Direct Interaction ◽  
Stable Complex ◽  
Extracellular Secretion ◽  
Triton X

ABSTRACT The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM ofVibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in anEscherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

scholarly journals Characterization of an adenosine 3′:5′-cyclic monophosphate phosphodiesterase from baker's yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour

1977 ◽  
Vol 163 (3) ◽  
pp. 467-476 ◽  
Author(s):  
J Londesborough
Keyword(s):  
Ionic Strength ◽  
Cyclic Amp ◽  
Microsomal Fraction ◽  
Gel Filtration ◽  
Potassium Phosphate ◽  
Apparent Molecular Weight ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Ph Optimum

1. The 3′:5′-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.

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