A Comparison of Physical Characteristics of Active Renin Isolated from Aorta, Plasma and Kidney of the Rat

1981 ◽  
Vol 61 (6) ◽  
pp. 671-678 ◽  
Author(s):  
J. D. Barrett ◽  
P. Eggena ◽  
J. F. Krall ◽  
M. P. Sambhi
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Proteolytic Enzymes ◽  
Rat Kidney ◽  
Kidney Tissue ◽  
Plasma Renin ◽  
Lower Molecular Weight ◽  
Aortic Tissue ◽  
Ph Optimum ◽  
Plasma Enzyme

1. Renin-like enzymes have been isolated from aorta, plasma and kidney tissue of the rat. The enzymes have been compared with respect to pH optima, molecular weight and isoelectric points. 2. Two distinct molecular-weight forms were isolated from plasma. The high-molecular-weight enzyme (mol. wt. > 150 000) appeared to be homogeneous with respect to isoelectric point (pI = 5.3), whereas the dominant lower-molecular-weight form (mol. wt. 44 000) demonstrated isoelectric heterogeneity. 3. The renin-like enzyme isolated from aortic tissue has mol. wt. 44 000 and also demonstrated isoelectric heterogeneity (pI = 5.0, 5.3). Plasma renin was significantly reduced after bilateral nephrectomy (30 h); however, the activity and relative proportions of the two aortic enzymes were not significantly altered. 4. Renin, isolated from kidney homogenates, either in the presence or absence of a variety of proteolytic enzyme inhibitors, had a significantly lower molecular weight (38 000) than the plasma enzyme but demonstrated a similar pattern of isoelectric heterogeneity. 5. in contrast with renin extracted from kidney, approximately 30% of the renin released from kidney cortical slices into Krebs-Ringer bicarbonate had mol. wt. 44 000. Only the 38 000 mol. wt. form was detected after storage of this medium and no change in total renin activity was evident. This suggests that the 38 000 mol. wt. form may be a methodological artifact due to the release of other proteolytic enzymes. 6. With homologous substrate, all fractionated enzymes showed a pH optimum between pH 7.0 and 7.5. 7. The present study indicates that the predominant form of renin released from rat kidney into blood has mol. wt. 44 000. The microheterogeneity of the plasma enzyme with respect to isoelectric point is similar to that of kidney renin. One form of renin (mol. wt. 44 000, pI = 5.0) may be common to kidney, plasma and aorta. The second aortic enzyme (pI = 5.3), however, may be of local origin.

Isolation and Purification to Apparent Homogeneity of 4,5-Dioxovalerate Aminotransferase from Scenedesmus obliquus Mutant C-2 A′

1988 ◽  
Vol 43 (7-8) ◽  
pp. 563-571 ◽  
Author(s):  
A. Kah ◽  
D. Dörnemann ◽  
H. Senger
Keyword(s):  
Molecular Weight ◽  
Green Alga ◽  
Isoelectric Point ◽  
Pyridoxal Phosphate ◽  
Scenedesmus Obliquus ◽  
Ph Optimum ◽  
Isolation And Purification ◽  
Apparent Homogeneity ◽  
Unicellular Green Alga ◽  
Glyoxylate Aminotransferase

In the present paper the purification of a specific 4,5-dioxovalerate transaminase from pigment mutant C-2 A′ of the unicellular green alga Scenedesmus obliquus to apparent homogeneity is described. The newly isolated enzyme ʟ-glutamate: 4,5-dioxovalerate aminotransferase is not identical with ʟ-alanine: 4,5-dioxovalerate aminotransferase (EC 2.6.1.43) and ʟ-alanine: glyoxylate aminotransferase (EC 2.6.1.44). A procedure for the purification is described and the resulting homogeneous protein is characterized by its Kᴍ-values for oxo-substrates and amino donors, its pyridoxal phosphate requirement, reversability of the catalysis, pH-optimum, isoelectric point and its molecular weight.

scholarly journals Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain

1979 ◽  
Vol 179 (3) ◽  
pp. 515-523 ◽  
Author(s):  
Thomas E. Knauer
Keyword(s):  
Molecular Weight ◽  
Rat Brain ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Supernatant Fraction ◽  
Ph Optimum ◽  
Factors Affecting ◽  
Chain Acyl ◽  
Coa Thioesters ◽  
Molecular Weight Form

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10μm-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl2, and 5,5′-dithiobis-(2-nitrobenzoic acid), and exhibit the same Km (1.8μm) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0°C), but there is no change in the Km with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10μm-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.

Separation and characterization of an α-glucosidase and maltase from Bacillus amyloliquefaciens

1985 ◽  
Vol 31 (8) ◽  
pp. 670-674 ◽  
Author(s):  
William M. Fogarty ◽  
Catherine T. Kelly ◽  
Sunil K. Kadam
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Growth Medium ◽  
Isoelectric Point ◽  
Bacillus Amyloliquefaciens ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Ph Optimum

A novel α-glucosidase and a maltase were isolated from Bacillus amyloliquefaciens. The formation of both enzymes was induced by trehalose, sucrose, or lactose in the growth medium. Trehalose is by far the most efficient inducer of both systems. The α-glucosidase and maltase were separated and purified by ion-exchange chromatography on DEAE Bio-Gel A. Purified α-glucosidase hydrolysed p-nitrophenyl-α-D-glucoside, isomaltose, and isomaltotriose but sucrose, maltose, or related saccharides were not attacked. β-Glucosides and polymeric glucosides were not degraded. The optimum temperature for α-glucosidase activity was 40 °C and its pH optimum was 5.3. The molecular weight and isoelectric point (pI) of the enzyme were 27 000 and 4.6, respectively. Purified maltase attacked maltose and sucrose, while maltotriose and melezitose were hydrolysed at slower rates and p-nitrophenyl-α-D-glucoside was not degraded. Other properties of the maltase were as follows: optimum temperature for activity, 30 °C; pH optimum, 6.5; molecular weight, 64 000; and pI, 4.7.

UDP-D-Xylose: Flavonol 3-O-Xylosyltransferase from Young Leaves of Euonymus alatus f. ciliato-dentatus

1991 ◽  
Vol 46 (11-12) ◽  
pp. 1003-1010 ◽  
Author(s):  
Nariyuki Ishikura ◽  
Zhi-qing Yang
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Isoelectric Point ◽  
Deae Cellulose ◽  
Sulfhydryl Groups ◽  
Hydroxyl Group ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Euonymus Alatus ◽  
Young Leaves

From the young leaves of Euonymus alatus f. ciliato-dentatus, a novel enzyme, UDP-D-xylose: flavonol 3-O-xylosyltransferase (F3XT), catalyzing the transfer of D-xylose from UDP-D-xylose to the 3 position of 3,5,7,4′-tetrahydroxyflavone (kaempferol), was detected and purified about 16-fold by precipitation with ammonium sulfate and DEAE-cellulose CC, by which F3XT was separated from two coexisting flavonol O-glucosyltransferases (FGT). Thus, F3XT was isolated as a soluble enzyme with a pH optimum of 7.0 in Tris-HCl buffer. The molecular weight of F3XT , which had an isoelectric point at pH 6.1, was estimated by elution from a column of Sephadex G-100 to be about 48 kDa. The activity of F3XT was stimulated by 14 mM 2-ME and strongly inhibited by 1 mAbstractм Cu2+, 1 mм Zn2+, and various re­ agents that react with sulfhydryl groups. Among the substrates tested for F3XT , kaempferol was the best. The Km values for kaempferol and UDP-xylose were determined to be 0.83 jim and 25 μM, respectively. F3XT mediated the transfer of xylose exclusively to the 3-hydroxyl group of kaempferol. Isorhamnetin, quercetin and fisetin also can function as xylosyl acceptor though less efficiently, but neither the 7-O-glucosides nor the 3-O-glucosides of kaempferol and quercetin were able to accept D-xylose. Dihydroflavonols were not xylosylated.

Mouse and rat plasma renin concentration and gene expression in (mRen2)27 transgenic rats

1998 ◽  
Vol 274 (5) ◽  
pp. H1450-H1456 ◽  
Author(s):  
Jürgen Bohlender ◽  
Joel Ménard ◽  
Oliver Edling ◽  
Detlev Ganten ◽  
Friedrich C. Luft
Keyword(s):  
Gene Expression ◽  
Rat Kidney ◽  
Plasma Renin ◽  
Rat Plasma ◽  
Transgenic Rat ◽  
Ribonuclease Protection Assay ◽  
Kinetic Assay ◽  
Ph Optimum ◽  
Plasma Renin Concentration ◽  
The Relationship

The (mRen2)27 transgenic rat [TGR(mRen2)27] is said to have low plasma levels of active renin. We used a direct radioimmunoassay (RIA) for mouse submaxillary renin, as well as an indirect enzyme-kinetic assay based on the generation of angiotensin I with modification of the pH optimum, to measure rat and mouse plasma renin activity (PRA), plasma renin concentration (PRC), and plasma prorenin in TGR before and after lisinopril. The relationship between rat PRC and %rat kidney extract was steepest at pH 6.0 and flat at pH 8.5, whereas the relationship between mouse PRC and purified mouse renin was steepest at pH 8.5 and flat at pH 6.0. Mouse PRC was highly correlated with direct RIA measurements ( r = 0.93). PRA before lisinopril was little influenced by pH, whereas the increase with lisinopril was greatest at pH 6.5. PRC before lisinopril was fourfold higher at pH 8.5 compared with that at pH 6.0. Lisinopril increased both PRC values but reversed the pH dependency. Prorenin was fourfold higher at pH 8.5 compared with that at pH 6.0 and decreased slightly with lisinopril. Renal renin concentration was higher at pH 6.0 than at pH 8.5. With lisinopril, renal renin concentration increased at both pH values. Mouse PRC was not changed by lisinopril. Ribonuclease protection assay showed both rat and mouse renin gene expression in the kidney, which increased with lisinopril. Thus TGR have circulating active rat and mouse renin and prorenin. The notion that TGR are a “low renin” model should be revised.

scholarly journals Substrate stabilization of the palmitoyl-coenzyme A hydrolase activity of rat submaxillary gland

1980 ◽  
Vol 187 (1) ◽  
pp. 269-272 ◽  
Author(s):  
T E Knauer ◽  
J J Gurecki ◽  
G R Knauer
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Submaxillary Gland ◽  
Hydrolase Activity ◽  
Lower Molecular Weight ◽  
Supernatant Fraction ◽  
Ph Optimum ◽  
Lower Molecular Weight Species ◽  
Substrate Stabilization ◽  
Coa Hydrolase

The long-chain acyl-CoA hydrolase (EC 3.1.2.2) activity of rat submaxillary salivary gland, found in the postmicrosomal supernatant fraction, has a pH optimum of 7.4. This hydrolase activity was found to be extremely labile, but inclusion of glycerol or the substrate palmitoyl-CoA in the preparations markedly stabilized the activity. Gel-filtration studies revealed multiple forms of the hydrolase, a lower-molecular-weight species of approx. 45 000 and a higher-molecular-weight species of approx. 130 000 observed when glycerol (20%, v/v) or palmitoyl-CoA (10 micro M) were included in the eluting buffer. This phenomenon is similar to that observed with the palmitoyl-CoA hydrolase of rat brain, except that there is no evidence that the higher-molecular-weight species of the hydrolase of submaxillary gland is generated by substrate-induced dimerization of the lower-molecular-weight species.

Handling of insulin by the isolated perfused rat kidney

1981 ◽  
Vol 240 (4) ◽  
pp. F288-F294 ◽  
Author(s):  
D. L. Maude ◽  
D. G. Handelsman ◽  
M. Babu ◽  
E. E. Gordon
Keyword(s):  
Molecular Weight ◽  
Glomerular Filtration Rate ◽  
Glomerular Filtration ◽  
Filtration Rate ◽  
Degradation Products ◽  
Rat Kidney ◽  
Kidney Tissue ◽  
Immunoreactive Insulin ◽  
Insulin Degradation ◽  
Organ Clearance

The organ clearance of insulin calculated from the rate of disappearance of immunoreactive insulin from the perfusate averages 0.76 ml.min-1.g kidneys wt-1, a value greater than the simultaneously measured glomerular filtration rate. Clearance does not fall when hormone concentration is as high as 7 X 10(-8) M (10,000 microunits/ml). Fifteen percent of the cleared insulin is excreted in the urine; the remainder is chemically modified and appears in the perfusate both as low molecular weight fragments and as high molecular weight species. In the process of clearing the hormone, kidney tissue accumulates both intact insulin and 125I-labeled insulin degradation products. the organ clearance of insulin is not curtailed when the glomerular filtration rate is sufficiently reduced (by lowering perfusate pressure) to cause urine flow to cease. Studies using hyperglycemic perfusates and kidneys taken from starving or streptozotocin-diabetic animals provided no evidence that the kidney plays a role in the regulation of plasma glucose by modulating the rate of insulin degradation.

Purification and properties of β-xylosidase from Penicillium wortmanni

1978 ◽  
Vol 56 (1) ◽  
pp. 43-50 ◽  
Author(s):  
F. Deleyn ◽  
M. Claeyssens ◽  
J. Van Beeumen ◽  
C. K. De Bruyne
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Ph Optimum ◽  
Purification Method ◽  
Acetone Precipitation ◽  
Purification And Properties ◽  
Tryptophan Residues ◽  
Rapid Inactivation ◽  
Inversion Mechanism

A purification method for an extracellular β-xylosidase (β-D-xyloside xylohydrolase, EC 3.2.1.37) induced in Penicillium wortmanni is described. It includes diafiltration, acetone precipitation, and hydroxylapatite chromatography. The enzyme has a molecular weight of about 100 000. Its pH optimum is at pH 3.3–4.0 and it is most stable at pH 5.0–6.0. Its isoelectric point is at pH 5.0. Sulfhydryl and histidine reagents are not inhibitory. The influence of added cations and anions is negligible. N-Bromosuccinimide oxidation of two to three tryptophan residues per molecule entails rapid inactivation. Glycon-specificity studies indicate strict requirements at C-2, C-3, C-4, and C-5, although α-L-arabinopyranosides are substrates. As the enzyme seems to hydrolyse xylooligosaccharides endwise, with retention of configuration in the reaction product, the enzyme is a true glycosidase, probably operating by a double-inversion mechanism.

scholarly journals STORED AND SECRETED RAT KIDNEY RENIN ISOFORMS DIFFER IN MOLECULAR WEIGHT AND ISOELECTRIC POINT. † 2179

1996 ◽  
Vol 39 ◽  
pp. 366-366
Author(s):  
Laura L Norling ◽  
Timothy M Smith ◽  
Julie R Ingelfinger
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Rat Kidney

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

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