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.

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.

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.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

scholarly journals The diphosphoinositide kinase of rat brain

1968 ◽  
Vol 106 (4) ◽  
pp. 791-801 ◽  
Author(s):  
M. Kai ◽  
J. G. Salway ◽  
J. N. Hawthorne
Keyword(s):  
Rat Brain ◽  
Kinase Activity ◽  
Ion Concentration ◽  
Supernatant Fraction ◽  
Thiol Groups ◽  
Ph Optimum ◽  
Adult Rat ◽  
Phosphatidylinositol Kinase ◽  
Adult Rat Brain ◽  
Ammonium Sulphate Fractionation

1. The supernatant fraction of adult rat brain contains a diphosphoinositide kinase. 2. Formation of triphosphoinositide by the enzyme in the presence of ATP and Mg2+ ions was shown with labelled ATP or labelled diphosphoinositide. 3. The kinase was also activated by Ca2+, Mn2+ and Co2+ ions, but to a smaller extent than by Mg2+ ions. 4. In the presence of optimum Mg2+ ion concentration the enzyme was inhibited by Ca2+ ions. 5. Activity did not depend on thiol groups and the pH optimum was 7·3. 6. The dialysed supernatant fraction had no diglyceride kinase activity and negligible phosphatidylinositol kinase activity. 7. Triphosphoinositide phosphomonoesterase was present but showed little activity under the conditions used to assay the kinase. 8. Diphosphoinositide kinase was purified by ammonium sulphate fractionation, ethanol treatment and chromatography on Sephadex G-200. 9. This purification removed much of the triphosphoinositide phosphomonoesterase.

scholarly journals Rat liver alcohol dehydrogenase. Purification and properties

1971 ◽  
Vol 125 (4) ◽  
pp. 1039-1047 ◽  
Author(s):  
M J Arslanian ◽  
E Pascoe ◽  
J G Reinhold
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Minor Component ◽  
Disc Electrophoresis ◽  
Supernatant Fraction ◽  
Liver Alcohol Dehydrogenase ◽  
A Minor

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.

scholarly journals Difference in Thrombolytic Effect between Higher and Lower Molecular Weight Forms of Urokinase

1977 ◽  
Author(s):  
T. Suyama ◽  
M. Nishida ◽  
Y. Iga ◽  
R. Naito
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Dissolution Time ◽  
Thrombolytic Activity ◽  
Lysis Time ◽  
Thrombolytic Effect ◽  
Approximate Molecular Weight ◽  
Made In

Urokinase (UK) from human urine has been widely used for thrombolytic therapy in Japan. However, commercially available preparations are not identical but consist of mainly two forms of UK with higher and lower molecular weight (H-UK and L-UK) . An attempt was made in this report to compare thrombolytic activity of H-UK with that of L-UK on artificial thrombi produced from human blood by a modification of Chandler’s loop method, which was somehow comparable to the situation in vivo. Two active forms of UK were purified from crude preparation by gel filtration. The approximate molecular weight of the H-UK was 54,000 and of the L-UK 34,000. The potency of UK was determined by “two-stage lysis time method” and expressed by International unit(lU). Thrombolytic activity measured by Chandler’s method was calculated as % lysis of the control thrombus that was formed in the abscence of UK.As a result, thrombus-dissolution time of H-UK was much shorter than that of L-UK. Furthermore, concentration of H-UK (IU/ml blood) necessary to induce 50% lysis was approximately one half lower than that of L-UK. The similiar results were obtained on artificial thrombi from the blood of dog, rat and rabbit. The data suggest that H-UK seems to be more effective on treatment of thromboembolicdisorders as compared to L-UK in terms of the same IU basis.

Partial purification and properties of an L-arabinose dehydrogenase from Azospirillum brasiliense

1983 ◽  
Vol 29 (2) ◽  
pp. 242-246 ◽  
Author(s):  
Norman J. Novick ◽  
Max E. Tyler
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Gel Filtration ◽  
Reducing Agent ◽  
Partial Purification ◽  
Ph Optimum ◽  
Glycine Buffer ◽  
Purification And Properties ◽  
Filtration Technique

An L-arabino-aldose dehydrogenase responsible for the oxidation of L-arabinose to L-arabino-γ-lactone has been purified 59-fold from L-arabinose grown cells of Azospirillum brasiliense. The dehydrogenase was found to be specific for substrates with the L-arabino-configuration at carbons 2, 3, and 4. Km values for L-arabinose of 75 and 140 μM were found with NADP and NAD as coenzymes, respectively. The enzyme had a pH optimum of 9.5 in glycine buffer and was stable when heated to 55 °C for 5 min. No enhancement of activity in the presence of any divalent cation or reducing agent tested was found. L-Arabinose dehydrogenase had a molecular weight of 175 000 as measured by the gel filtration technique.

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.

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.

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