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.

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.

Multiple forms of immunoreactive renin in human adrenocortical tumour tissue from patients with primary aldosteronism

1987 ◽  
Vol 72 (6) ◽  
pp. 699-704 ◽  
Author(s):  
Kenji Mizuno ◽  
Motoko Ojima ◽  
Shigeatsu Hashimoto ◽  
Makio Tani ◽  
Susumu Niimura ◽  
...  
Keyword(s):  
Rat Plasma ◽  
Biochemical Properties ◽  
Zona Glomerulosa ◽  
Multiple Forms ◽  
Angiotensin I ◽  
Isoelectric Points ◽  
Optimum Ph ◽  
Glomerulosa Cells ◽  
Adrenocortical Tumour ◽  
Zona Glomerulosa Cells

1. There is increasing evidence which suggests that the adrenal gland contains the renin–angiotensin cycle. The localization of renin has been reported to be mainly in the zona glomerulosa rather than the fasciculata medullary portion. In the present study we have investigated extracts from aldosteronomas (n = 3), which are believed to derive from the zona glomerulosa cells. In addition, we have attempted to characterize the biochemical properties of the adrenal renin. 2. Sizable quantities of renin-like activity (32.0 ± 7.7 ng of angiotensin I generated h−1 mg−1 of protein, mean ± SEM) were detected in the extracts. This renin-like activity was inhibited by anti-renin antibody raised against pure renin (mean, 95% of the total renin-like activity), indicating that it was not due to the non-specific action of proteases such as cathepsin D. 3. The optimum pH of the tissue renin-like enzyme was 6.0 for rat plasma substrate. Differences were found, however, in the molecular mass (36000, 37000, 44000 and 48000), binding to concanavalin A and isoelectric points (4.40, 4.68 and 5.00). 4. These results confirm the existence of specific renin in aldosteronoma. Renin microheterogeneity could be evidence for local production of the enzyme.

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.

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 A comparison of the properties of renin isolated from pig and rat kidney

1976 ◽  
Vol 155 (2) ◽  
pp. 317-323 ◽  
Author(s):  
M Lauritzen ◽  
J J Damsgaard ◽  
I Rubin ◽  
E Lauritzen
Keyword(s):  
Isoelectric Focusing ◽  
Rat Kidney ◽  
Substrate Affinity ◽  
Acid Activation ◽  
Renin Substrate ◽  
Crossed Immunoelectrophoresis ◽  
Inactive Form ◽  
Isoelectric Points ◽  
Michaelis Constants ◽  
Rat Kidneys

1. On isoelectric focusing, renin from rat kidneys showed three activity peaks with pI values at pH 5.0, 5.2 and 5.4 after a purification procedure involving differential centrifugation, acidification, chromatography on Sephadex G-75 and dialysis. 2. The preparation (purified 140-fold) was compared with a crude kidney extract in the absence and presence of 3 M-urea by isoelectric focusing. The pattern of activity distribution was confirmed by these experiments and the content of isoenzymes in the three groups calculated. 3. Pig renin was prepared and compared with rat renin with regard to molecular weight, acid activation, behaviour on isoelectric focusing, immunogenicity and substrate affinity. 4. Extracts of rat kidney contained multiple forms of renin with mol.wt. between 39000and 42000, whereas active pig renin had an approximate mol.wt. of 40000. Acidification of rat renal extracts did not increase the activity of renin, indicating the absence of an inactive form of renin in rat kidneys, whereas pig renin was activated by this procedure. Pig renin has isoelectric points at pH 4.6, 4.8, 5.05 and 5.2, significantly lower than for rat renin. The isoenzymes from the two species had no antigenicity in common, as shown by crossed immunoelectrophoresis or rocket immunoelectrophoresis. 5. The Michaelis constants for pig and rat renin were in the same range, 1 × 10(-6) M, when rat renin substrate was used. The relative content of rat isoenzyme with pI in the pH ranges 4.9-5.1, 5.1-5.3 and 5.3-5.5 was approx. 20, 27 and 53% respectively. Purified pig renin prepared in two different ways had isoenzymes with pI in the pH regions 4.5-4.7, 4.7-4.9, 4.9-5.05 and 5.05-5.20 in the approximate proportions 14, 24, 28 and 29%.

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.

scholarly journals Anomalous behaviour of yeast isocitrate dehydrogenase during isoelectric focusing

1972 ◽  
Vol 129 (5) ◽  
pp. 1125-1130 ◽  
Author(s):  
John A. Illingworth
Keyword(s):  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Isocitrate Dehydrogenase ◽  
Band Pattern ◽  
Anomalous Behaviour ◽  
Multiple Forms ◽  
Isoelectric Points ◽  
The Mean ◽  
Crude Material

Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ‘isoenzymes’. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.

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.

Trypsin activation of inactive renin: plasma blanks and angiotensin I radioimmunoassay

1991 ◽  
Vol 69 (9) ◽  
pp. 1315-1320 ◽  
Author(s):  
Jack D. Barrett ◽  
Peter Eggena
Keyword(s):  
Substrate Concentration ◽  
Rat Plasma ◽  
Direct Proportion ◽  
Trypsin Treatment ◽  
Angiotensin I ◽  
Renin Substrate ◽  
Active Renin ◽  
Normal Plasma ◽  
Inactive Renin ◽  
Significant Bearing

Divergent conclusions exist as to whether inactive renin is present in nephrectomized rat plasma. A major factor contributing to this conflict may be related to significant changes in the "plasma blank" when trypsin-treated plasma is subjected to angiotensin I (AI) radioimmunoassay (RIA). In normal, but not nephrectomized rat plasma, AI-like substances are present in direct proportion to active renin. These substances are destroyed by trypsin. However, trypsin generates additional AI-like material, in both normal and nephrectomized rat plasma. This material, which is present in proportion to the renin substrate concentration, does not appear to be tetradecapeptide (TDP). In normal plasma, however, exogenous TDP is converted to AI in proportion to the active renin concentration and AI generation from TDP is increased by activation of inactive renin. However, in nephrectomized rat plasma, no AI generation from TDP was evident either before or after trypsin treatment. The coincident tryptic generation of a substance that quenches the levels of AI detected by RIA, combined with significant changes in the levels of endogenous and trypsin generated AI-like substances, may have significant bearing on the measured levels of inactive renin.Key words: prorenin, nephrectomy, angiotensin I radioimmunoassay, rat, plasma blanks.

scholarly journals Multiple forms of γ-butyrobetaine hydroxylase (EC 1.14.11.1)

1984 ◽  
Vol 223 (1) ◽  
pp. 119-127 ◽  
Author(s):  
S Lindstedt ◽  
I Nordin
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Polyacrylamide Gels ◽  
Multiple Forms ◽  
Bacterial Enzyme ◽  
Isoelectric Points ◽  
Liver And Kidney

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.

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