Fractionation and further characterization of granulocytic and monocytic alpha-naphthyl acetate (ANAE) esterases.

1984 ◽  
Vol 32 (6) ◽  
pp. 579-584 ◽  
Author(s):  
C S Scott ◽  
D Hough ◽  
A G Bynoe ◽  
D B Jones ◽  
B E Roberts
Keyword(s):  
Cell Differentiation ◽  
Isoelectric Focusing ◽  
Myeloid Cell ◽  
The Other ◽  
Monocytic Differentiation ◽  
Isoelectric Points ◽  
Monocytic Lineage ◽  
Naphthyl Acetate ◽  
Nonspecific Esterases

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.

scholarly journals Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis.

1978 ◽  
Vol 26 (5) ◽  
pp. 382-390 ◽  
Author(s):  
C C Phillips Burnett ◽  
W A Anderson ◽  
R Rüchel
Keyword(s):  
Molecular Weight ◽  
Isoelectric Focusing ◽  
Large Subunit ◽  
The Other ◽  
Two Dimensional ◽  
Neuraminidase Treatment ◽  
Gradient Electrophoresis ◽  
Isoelectric Points ◽  
Uterine Fluid

Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.

Isoelectric focusing of insulin, 125I-insulin and the 125I-insulin-antibody complex

1979 ◽  
Vol 44 (6) ◽  
pp. 1828-1834
Author(s):  
Asja Šiševa ◽  
Jiřina Slaninová ◽  
Tomislav Barth ◽  
Stephan P. Ditzov ◽  
Luben M. Sirakov
Keyword(s):  
Isoelectric Focusing ◽  
Polyacrylamide Gel ◽  
The Other ◽  
Insulin Antibody ◽  
Isoelectric Points ◽  
Antibody Complex ◽  
And Storage ◽  
Acid Region ◽  
Three Components

Isoelectric focusing on polyacrylamide gel columns of three native crystalline commercial preparations of insulin and 125I-labelled insulin was carried out. All the compounds studied contained three components of different isoelectric points. The largest fraction, having pI 5.60 ± 0.05, was common to all preparations. The other two fractions were situated in the acid region of pH between pI 4.5 and 5.2. The presence of these fractions is explained by the contamination of crystalline insulins by proinsulin and by the formation of des-amido derivatives during the dissolving and storage of insulin samples, and, in case of labelled insulin, also by the presence of heavily iodinated insulin and contaminating components. The isoelectric focusing of the complex 125I-insulin-antibody showed a peak of radioactivity having pI 6.15 ± 0.05.

Resolution of rat renin substrates by isoelectric focusing

1977 ◽  
Vol 55 (8) ◽  
pp. 869-875 ◽  
Author(s):  
A. A. Faiers ◽  
A. Y. Loh ◽  
D. H. Osmond
Keyword(s):  
Isoelectric Focusing ◽  
Substrate Concentration ◽  
Rat Plasma ◽  
The Other ◽  
Multiple Forms ◽  
High Substrate Concentration ◽  
Broad Distribution ◽  
Renin Substrate ◽  
Isoelectric Points ◽  
Different Sources

Pooled plasmas from normal or binephrectomized rats and perfusates of isolated livers were used as sources of renin substrate for isoelectric focusing. After desalting, preliminary fractionation (plasma only), and concentration, the preparations were focused in a pH 3–10 gradient on 20-cm glass plates layered with Sephadex slurry. The pH 4–6 region, containing all the substrate, was scraped from this plate and refocused in a pH 4–6 gradient. Substrate content of 1-cm strips of slurry from half of the plate was determined by both radioimmunoassay and bioassay of angiotensin resulting from incubation with added renin. Corresponding strips from the other half of the plate were incubated without renin as a control for any preformed angiotensin. The asymmetry and broad distribution (pH 4–5) of substrate from different sources suggested the existence of more than one form. Higher resolution achieved by using the high substrate concentration of postnephrectomy plasma and 0.5-cm strips of slurry on 20-cm or 40-cm plates revealed peaks and shoulders of substrate activity. Our data suggest that multiple forms of substrate are synthesized by the liver and circulate in plasma. Postnephrectomy rat plasma appears to contain relatively more substrate(s) with higher isoelectric points than in normal plasma, possibly an accumulation of forms ordinarily degraded by endogenous renal renin.

scholarly journals Formation and properties of flavoprotein-cytochrome hybrids by recombination of subunits from different species

1985 ◽  
Vol 231 (2) ◽  
pp. 383-387 ◽  
Author(s):  
S C Koerber ◽  
D J Hopper ◽  
W S McIntire ◽  
T P Singer
Keyword(s):  
Catalytic Activity ◽  
Steady State ◽  
Isoelectric Focusing ◽  
Dissociation Constants ◽  
The Other ◽  
High Dilution ◽  
Steady State Kinetic ◽  
Isoelectric Points ◽  
State Kinetic ◽  
Enzyme Species

p-Cresol methylhydroxylases from four different pseudomonads differ in their isoelectric points and, to a lesser extent, in Mr values and substrate specificity. The enzymes from three species were isolated in homogeneous form, then resolved into their flavoprotein and cytochrome subunits, and the subunits were recombined to yield the nine possible hybrids (i.e. three intraspecies and six interspecies). The resulting flavocytochromes showed extensive similarities in steady-state kinetic parameters and in the dissociation constants of their subunits. Evidence is also presented that a fourth type of p-cresol methylhydroxylase, from Pseudomonas putida (N.C.I.B. 9869, form ‘B’), the subunits of which cannot be isolated by the isoelectric focusing technique used to separate the subunits of the other flavocytochromes, nevertheless dissociates slowly at high dilution. The dissociation is reflected by a decline of catalytic activity with time. This process for the ‘B’ enzyme is prevented by the presence of substrate or an excess of a cytochrome subunit isolated from another enzyme species. Incubation of the dissociated subunits with p-cresol brings about extensive, albeit incomplete, re-association and regeneration of activity.

scholarly journals Nonspecific esterase isozymes that focus at identical isoelectric points are kinetically analogous.

1987 ◽  
Vol 35 (2) ◽  
pp. 275-276
Author(s):  
S R Choudhury
Keyword(s):  
Isoelectric Focusing ◽  
Soluble Fraction ◽  
Rat Kidney ◽  
Kinetic Properties ◽  
Nonspecific Esterase ◽  
Kinetic Behavior ◽  
Esterase Isozymes ◽  
Isoelectric Points ◽  
Nonspecific Esterases

Isoelectric focusing (IEF) of soluble nonspecific esterases of rat kidney and testis exhibits an identical array of organophosphate-resistant cathodal isozymes. To ascertain whether such isozymes that focus at the same pI are also kinetically analogous, two isozymes, both focused at pI 7.2, were isolated, one from each organ, by elution from cut-out, unstained gel segments. Although the esterases of the whole soluble fraction of kidney and testis exhibited different kinetic properties and organophosphate susceptibility, no differences were observed with regard to the isozymes. Therefore, because of similar electrophoretic and kinetic behavior, the two isozymes can be regarded as phenotypic expressions of similar genetic products.

scholarly journals Characterization of isoelectric subspecies of asialo-β2-glycoprotein I

1989 ◽  
Vol 260 (2) ◽  
pp. 531-534 ◽  
Author(s):  
A Gries ◽  
J Nimpf ◽  
H Wurm ◽  
G M Kostner ◽  
T Kenner
Keyword(s):  
Western Blot ◽  
Isoelectric Focusing ◽  
Plasma Samples ◽  
Neuraminidase Treatment ◽  
Glycoprotein I ◽  
Isoelectric Points ◽  
Cation Exchange Column ◽  
Beta 2 ◽  
Native Plasma

Isoelectric focusing of purified beta 2-glycoprotein I (beta 2-G-I) revealed five major bands with isoelectric points (pI) between 5.1 and 6.1. Neuraminidase treatment decreased the number of bands to two (pI 8.0 and 8.2). The two asialo subfractions of beta 2-G-I were purified by cation-exchange column chromatography. The more basic isoform II was found to have a higher content of lysine. Western-blot analysis of different plasma samples confirmed the heterogeneity of beta 2-G-I in plasma. Plasma treated with neuraminidase showed two bands irrespective of the number of isoforms as well as of the concentration in native plasma. This led us to the conclusion that human plasma beta 2-G-I consists of two isoproteins that are sialylated to different extents.

Purification and characterization of two forms of Cu/Zn-superoxide dismutase from bovine erythrocytes by preparative isoelectric focusing

1981 ◽  
Vol 46 (9) ◽  
pp. 2140-2145 ◽  
Author(s):  
Hana Kovářová ◽  
Jan Šimša ◽  
Josef Křížala
Keyword(s):  
Superoxide Dismutase ◽  
Isoelectric Focusing ◽  
Visible Region ◽  
Alkaline Media ◽  
Ultraviolet Region ◽  
Purification And Characterization ◽  
Isoelectric Points ◽  
Relative Molecular Weight ◽  
Bovine Erythrocytes

Two forms of Cu/Zn-superoxide dismutase with isoelectric points (pI) 6.3 and 5.2 were isolated from bovine erythrocytes by preparative isoelectric focusing. Both forms show a relative molecular weight of 32 000 daltons corresponding to the value reported for the monomer molecule. From spectral analysis the maximum in the ultraviolet region of the spectrum (276 nm) is identical for both forms of superoxide dismutase whereas the maxima in the visible region are different (for the pI 5.2 form the maximum lies at 405 nm and for the pI 6.3 form at 695 nm). The different migration of the enzymatically active zones toward the anode during electrophoresis in alkaline media corresponds to their different isoelectric points.

Penicillocarboxypeptidase-S, a Nonspecific SH-Dependent Exopeptidase

1972 ◽  
Vol 50 (12) ◽  
pp. 1297-1310 ◽  
Author(s):  
S. R. Jones ◽  
T. Hofmann
Keyword(s):  
Isoelectric Focusing ◽  
Culture Medium ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
The Other ◽  
Ph Optimum ◽  
Penicillium Janthinellum ◽  
Diisopropyl Phosphorofluoridate ◽  
Isoelectric Points ◽  
Inhibition Studies

An extracellular carboxypeptidase with a pH optimum of between 4 and 5 has been isolated from the culture medium of Penicillium janthinellum. Like penicillopepsin, this enzyme is only released when the stationary phase of growth is reached. The enzyme has been purified by affinity chromatography on phenylbutylamine-Sepharose followed by isoelectric focusing. The enzyme was found to be present in two forms with isoelectric points of 3.70 and 3.77. It has a molecular weight of about 48 000 and is inhibited by 10−7 M p-hydroxymercury benzoic acid. It is not inhibited by the metal chelators EDTA, o-phenanthroline, and 8-hydroxyquinoline, or by diisopropyl phosphorofluoridate. The presence of a single cysteine residue has been demonstrated by labelling the enzyme with 14C-iodoacetic acid. The action of the enzyme on glucagon, the S-sulfo-B-chain of insulin, and on penicillopepsin at pH 4.7 shows that it is a nonspecific carboxypeptidase and releases C-terminal proline, lysine, and arginine, as well as the other amino acids. Glycine appears to be the slowest residue to be released. Carbobenzoxy-L-glutamyl-L-tyrosine, the substrate used for routine assays, is hydrolyzed very rapidly. The enzyme also slowly hydrolyzed Leu–Phe, and splits glycine from Leu–Gly–Gly.A second enzyme with carbobenzoxy-L-glutamyl-L-tyrosine activity is present in the growth medium. It has an isoelectric point of 4.72 on an isoelectric focusing column. Preliminary inhibition studies of a partially purified preparation suggest that it differs from the other enzyme.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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