Characteristics of a Renin-Binding Substance for the Conversion of Renin into a Higher-Molecular-Weight Form in the Dog

1979 ◽  
Vol 57 (4) ◽  
pp. 345-350 ◽  
Author(s):  
Minoru Kawamura ◽  
Fumihiko Ikemoto ◽  
Susumu Funakawa ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Filtration Fraction ◽  
Acid Ph ◽  
Binding Substance ◽  
Molecular Weight Form

1. Renal cortical homogenates of the dog were subjected to sieve separation, a Nucleopore Filter being used to separate the renin granules. 2. The molecular weight of renin in the granules was estimated to be about 40 000 by gel filtration. Renin was converted into a higher-molecular-weight form (60 000) by mixing with cytosol in the presence of sodium tetrathionate, a thiol inhibitor. 3. When cytosol was pretreated with acid (pH 30) or heating (100°C), the molecular-weight conversion did not occur. 4. Cytosol was separated into three parts by gel filtration. Fraction A included substances with a molecular weight of over 47 000, fraction B from 47 000 to 32 000, and fraction C from 32 000 to 15 000. The mixture of renin in the granules with fraction A and sodium tetrathionate resulted in the formation of a higher-molecular-weight form of the enzyme, but no change in molecular weight was detected when renin was mixed with fractions B or C and sodium tetrathionate.

Preliminary Evidence for the Conversion of Dog Renin into a Higher—Molecular—Weight Form by Cold Storage

1980 ◽  
Vol 58 (6) ◽  
pp. 451-456 ◽  
Author(s):  
Minoru Kawamura ◽  
Fumihiko Ikemoto ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
Cold Storage ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Molecular Size ◽  
Preliminary Evidence ◽  
Concomitant Increase ◽  
Binding Substance ◽  
Dog Kidney ◽  
Molecular Weight Form

1. A soluble fraction of renal cortical homogenate (cytosol) and renin extracted from isolated renin granules of the dog kidney were kept at 0°C. 2. Although the molecular weight of the renin in the cytosol on day 1 was estimated to be about 40 000 by gel filtration, the renin was completely converted into a higher—molecular—weight form (60 000) by day 7. The renin in the granules kept its molecular size of 40 000 during cold storage. 3. This type of molecular—weight conversion could be performed without protease inhibitors. 4. Dithiothreitol neither inhibited the conversion into the higher—molecular—weight form of renin during cold storage nor led to a reduction in the molecular weight of renin, although the oxidation of thiol groups has been proposed as the mechanism for the molecular—weight conversion of renin. 5. Keeping a mixture of renin from the granules and crude renin—binding substance at 0°C for 7 days resulted in the conversion of the renin into the higher—molecular—weight form, indicating that the renin—binding substance we have previously described is required for the conversion during cold storage. 6. Acidification caused the higher—molecular—weight form of renin formed in the cytosol to change to the lower—molecular—weight form, with a concomitant increase in activity of about 50%.

scholarly journals High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates

1977 ◽  
Author(s):  
K. A. Rickard ◽  
T. Exner ◽  
H. Kronenberg
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Human Antibody ◽  
High Molecular Weight Material ◽  
Coagulant Activity ◽  
Molecular Weight Form

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

Immunoaffinity Chromatography of Canine High-Molecular-Weight Renin: Partial Purification and Characterization

1983 ◽  
Vol 65 (2) ◽  
pp. 117-120 ◽  
Author(s):  
Fumihiko Ikemoto ◽  
Victor J. Dzau ◽  
Edgar Haber ◽  
Kazuo Takaori ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Immunoaffinity Chromatography ◽  
Partial Purification ◽  
Low Molecular Weight ◽  
Specific Method ◽  
Disulphide Bonds ◽  
Binding Substance ◽  
Molecular Weight Form

1. Canine high-molecular-weight renin (mol. wt. 60 000) is believed to be a complex of renin (low-molecular-weight form, mol. wt. 40 000) and renin-binding substance. The immunocross-reactivity of high-molecular-weight renin and low-molecular-weight renin was demonstrated by using antibodies specific to low-molecular-weight renin. 2. Immunoaffinity chromatography with renin-specific antibodies coupled to Sepharose provided a simple and specific method for isolation of high-molecular-weight renin. High-molecular-weight renin with a specific activity of 137 600 ng of ANG I h−1 mg−1 of protein (19.6 Goldblatt units/mg of protein) was obtained. 3. This high-molecular-weight renin was stable in dithiothreitol (25 mmol/l), suggesting that disulphide bonds may not be involved in the binding mechanism between low-molecular-weight renin and renin-binding substance. 4. However, exposure to low pH (3.0) resulted in conversion of high-molecular-weight renin into the low-molecular-weight form.

scholarly journals Studies on the appearance of a hepatic copper-binding protein in normal and zinc-deficient rats

1976 ◽  
Vol 36 (1) ◽  
pp. 101-112 ◽  
Author(s):  
I. Bremner ◽  
N. T. Davies
Keyword(s):  
Molecular Weight ◽  
Metal Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Low Molecular Weight ◽  
Copper Binding ◽  
Hepatic Copper ◽  
Copper Binding Protein ◽  
Molecular Weight Form

1. A study has been made by gel-filtration techniques of the soluble copper- and zinc-binding proteins in rat liver after both intraperitoneal injection of Cu and dietary Cu supplementation.2. Liver Cu and Zn concentrations increased after injection of Cu, both metals accumulating in the cytosol, mainly in a fraction with an apparent molecular weight of (about 12 000)3. When Zn-deficient rats were injected with Cu, there was little change in liver Zn concentration and the occurrence of Cu in the low-molecular-weight form (about 12 000) was more transient. At most periods after injection, Cu accumulated mainly in a fraction with a molecular weight greater than 65 000.4. When the rats were Cu-loaded by dietary supplementation, virtually no Cu or Zn was found in the low-molecular-weight form in Zn-deficient rats, although they were found in the Zn-supplemented animals.5. The results suggest that Zn is essential for the accumulation of Cu in this form, but not for Cu to stimulate production of the metal-binding protein by a process requiring active protein synthesis.

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.

Renin Precursor and Its Activation Mechanism in Hog Kidney

1980 ◽  
Vol 59 (s6) ◽  
pp. 21s-24s ◽  
Author(s):  
Kazuo Murakami ◽  
Saori Takahashi ◽  
Shigehisa Hirose ◽  
Yukio Takii ◽  
Tadashi Inagami
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Activation Mechanism ◽  
Net Charge ◽  
Isoelectric Points ◽  
Binding Substance ◽  
Inactive Renin ◽  
Hog Kidney

1. A completely inactive renin was isolated from hog kidney extract by affinity chromatography on pepstatin-aminohexyl-Sepharose and on an Affi-Gel Blue column. 2. This inactive renin had a molecular weight of 43 000 ± 1500 as determined by gel filtration on Ultrogel AcA 44. Upon activation with trypsin, its molecular weight fell to 41 000 ± 1400. 3. The inactive renin lacked the ability to bind renin-binding substance whereas trypsin-activated renin was able to bind the renin-binding protein and to form high-molecular-weight renin. 4. Chymotrypsin as well as trypsin could activate the inactive renin although less effectively. 5. The active renins generated from the inactive renin by the action of different proteolytic enzymes differed in their net charge, reflecting the specificities of the proteases used; the isoelectric points of the native, the trypsin-activated and the chymotrypsin-activated forms of renin occurred at pH 5.3, 5.1 and 4.8 respectively.

scholarly journals Schwann cell proliferation in vitro is under negative autocrine control.

1990 ◽  
Vol 111 (6) ◽  
pp. 2663-2671 ◽  
Author(s):  
D Muir ◽  
S Varon ◽  
M Manthorpe
Keyword(s):  
Molecular Weight ◽  
Schwann Cell ◽  
Molecular Mass ◽  
Conditioned Medium ◽  
Schwann Cells ◽  
Cell Cultures ◽  
Antiproliferative Activity ◽  
Gel Filtration ◽  
Molecular Weight Form

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen-stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol

1979 ◽  
Vol 57 (6) ◽  
pp. 605-612 ◽  
Author(s):  
Patrick C. Choy ◽  
S. Blake Farren ◽  
Dennis E. Vance
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Neutral Lipids ◽  
Gel Filtration ◽  
Fold Increase ◽  
High Cholesterol Diet ◽  
Liver Cytosol ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Molecular Weight Form ◽  
Rat Liver Cytosol

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 °C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.

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