scholarly journals Isolation of Two Distinct Activator Proteins For Lipoprotein Lipase from Ovine Plasma

1987 ◽  
Vol 40 (3) ◽  
pp. 235
Author(s):  
RK Tume ◽  
RF Thornton ◽  
OW Johnson
Keyword(s):  
Lipoprotein Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Significant Inhibition ◽  
Activator Proteins ◽  
Human Apolipoprotein ◽  
Apolipoprotein C ◽  
Maximum Activation ◽  
Hydrolysis Of ◽  
Low Molecular Weight Proteins

Two distinct activator proteins for lipoprotein lipase (LPL) have been isolated in approximately equal amounts from ovine plasma. These low molecular weight proteins were readily separated from each other on the basis of size and charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated proteins of Mr about 8000 and 5000, with pI in urea-containing gels of about 5�1 and 4�8 respectively. Each of the ovine activator proteins was as effective as human apolipoprotein C-II (apo C-II) in activating LPL, with I JLg/ml giving near to maximum activation, and in lowering the apparent Km of LPL for triolein substrate. As the ratio of activator to triolein increased from 0�16 to 5� 2 (JLg/mg) the apparent Km fell from about O' 5 to 0 �18 mM. Whereas human apo C-II and the two ovine activators were equally effective in stimulating the hydrolysis of triolein, differences were observed between the human and ovine activators when p-nitrophenylbutyrate was used as substrate. Unlike human apo C-II, which produced significant inhibition of p-nitrophenylbutyrate hydrolysis, the ovine activators were without effect. This suggests that the interaction between the ovine activators and LPL is different from that of human apo C-II.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis

1982 ◽  
Vol 152 (2) ◽  
pp. 616-625
Author(s):  
D J Mancuso ◽  
T H Chiu
Keyword(s):  
Thin Layer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Lipoteichoic Acid ◽  
Molar Ratio ◽  
Streptococcus Sanguis ◽  
Chromatographic Profile ◽  
Hydrolysis Of ◽  
Cellulose Column

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.

Changes in total and individual proteins during drying and ruminal fermentation of alfalfa

2005 ◽  
Vol 2005 ◽  
pp. 198-198
Author(s):  
A. A. Sadeghi ◽  
P. Shawrang ◽  
M. Moradi ◽  
A. Nikkhah
Keyword(s):  
Crude Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Plant Proteins ◽  
Ruminal Fermentation ◽  
Protein Nitrogen ◽  
Sds Page ◽  
Total Crude Protein ◽  
Hydrolysis Of

Proteolysis within plant cells occurs during wilting and drying. Changes in plant proteins during those periods usually are monitored by measurement of total crude protein and non protein nitrogen. Alternatively, changes in concentrations of individual proteins can be measured. Plants are composed of an array of different proteins. Electrophoresis can be used to separate these proteins and has been used to study effects of wilting and ensiling on proteins of some forages (Grum et al., 1991). Electrophoresis also has been used in the study of ruminal hydrolysis of oilseed meals proteins (Sadeghi et al., 2004). Most of the experiments designed to use electrophoresis to study protein metabolism in forages and ruminants have been qualitative. The main objective of this study was to determine whether sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry could be used to monitor quantitatively the changes in alfalfa protein composition during wilting, drying and ruminal exposure.

scholarly journals A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum)

1976 ◽  
Vol 157 (3) ◽  
pp. 745-751 ◽  
Author(s):  
P Smirnoff ◽  
S Khalef ◽  
Y Birk ◽  
S W Applebaum
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Polyacrylamide Gel ◽  
Molar Ratio ◽  
Chymotrypsin Inhibitor ◽  
Trypsin Inhibitory Activity ◽  
Hydrolysis Of

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.

Substitution of SER61 → GLY61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase

1986 ◽  
Vol 39 (4) ◽  
pp. 341-346 ◽  
Author(s):  
A. Balasubramaniam ◽  
R.A. Demel ◽  
R.F. Murphy ◽  
J.T. Sparrow ◽  
R.L. Jackson
Keyword(s):  
Lipoprotein Lipase ◽  
Human Apolipoprotein ◽  
Apolipoprotein C

NMR Structure of Human Apolipoprotein C-II in the Presence of Sodium Dodecyl Sulfate†

Biochemistry ◽  
2001 ◽  
Vol 40 (18) ◽  
pp. 5414-5421 ◽  
Author(s):  
Christopher A. MacRaild ◽  
Danny M. Hatters ◽  
Geoffrey J. Howlett ◽  
Paul R. Gooley
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Nmr Structure ◽  
Human Apolipoprotein ◽  
Apolipoprotein C ◽  
Dodecyl Sulfate

Method for measuring the concentration of urinary proteins according to their molecular size category.

1976 ◽  
Vol 22 (5) ◽  
pp. 667-672 ◽  
Author(s):  
A J Pesce ◽  
A Hsu ◽  
C Kornhauser ◽  
K Sethi ◽  
B S Ooi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Low Molecular Weight ◽  
Tubular Proteinuria ◽  
Equal Concentration ◽  
Glomerular Proteinuria ◽  
Low Molecular Weight Proteins

Abstract We combined the use of a concentrating device (Minicon) and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to semi-quantitate the concentration of (a) the collective low-molecular-weight proteins and (b) of albumin excreted in the urine of patients after renal transplantation. Analytical recovery of many serum proteins from samples concentrated 100-fold in the Minicon apparatus was about 70%. It was possible to examine many urine samples by polyacrylamide gel electrophoresis after concentration with this device. The reproducibility (CV) of the technique was on the order of 20% when albumin and low-molecular-weight protein were in about equal concentration. The method was adequate to differntiate glomerular and tubular proteinuria, because in glomerular proteinuria the ratio of albumin to low-molecular-weight proteins is about 20/1, whereas in tubular proteinuria the ratio is about 1/1.

scholarly journals Early Renal Involvement in Cats with Natural Feline Morbillivirus Infection

Animals ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 828 ◽  
Author(s):  
Paolo Emidio Crisi ◽  
Francesco Dondi ◽  
Eliana De Luca ◽  
Morena Di Tommaso ◽  
Kateryna Vasylyeva ◽  
...  
Keyword(s):  
Specific Gravity ◽  
Renal Damage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Renal Involvement ◽  
Sodium Dodecyl ◽  
Urine Specific Gravity ◽  
Creatinine Concentration ◽  
Urine Creatinine ◽  
Sds Page ◽  
Low Molecular Weight Proteins

Feline morbillivirus (FeMV) is a newly discovered paramyxovirus infecting domestic cats and its role in the pathogenesis of feline chronic kidney disease (CKD) has been suggested, however not confirmed. The primary aim of the study was to evaluate the renal damage associated with FeMV infection in cats. In this retrospective study, clinical and clinicopathological data were compared among 14 FeMV naturally infected, 21 CKD and 22 healthy cats. FeMV positive cats had serum chemistry analytes and main urine chemistry results similar to the healthy subjects. FeMV positive cats had significantly decreased urine specific gravity (median 1054, range 1022–1065) and urine creatinine (median 227.23 mg/dL, range 83.02–489.75) when compared with healthy cats (median 1067, range 1040–1080, p < 0.001; median 406.50 mg/dL, range 195.32–575.58; p < 0.001, respectively). Urine protein:creatinine ratio (UPC) results of FeMV and CKD were not different (median 0.20, range 0.08–1.03; median 0.23, range 0.10–0.80, respectively), however UPC results were significantly increased in both groups, if compared with healthy cats (median 0.1, range 0.04–0.250, p < 0.01). Based on clinical data, serum creatinine concentration, urine specific gravity and UPC results, CKD was suspected by clinicians in 3/14 FeMV cats. Urine protein sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10/13 (77%) FeMV cats indicated a tubular pattern, with a decrease of uromodulin and an increase in the number and intensity of low molecular weight proteins. FeMV infection can be associated with different grades of renal dysfunction ranging from mild tubular proteinuria with less concentrated urine to azotemia in cats younger than those typically affected by CKD.

scholarly journals SYNTHESIS OF GLYCOPROTEINS IN A SINGLE IDENTIFIED NEURON OF APLYSIA CALIFORNICA

1974 ◽  
Vol 61 (3) ◽  
pp. 649-664 ◽  
Author(s):  
Richard T. Ambron ◽  
James E. Goldman ◽  
Elizabeth Barnes Thompson ◽  
James H. Schwartz
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Mild Acid Hydrolysis ◽  
Giant Neuron ◽  
Electrophoretic Patterns ◽  
Identified Neuron ◽  
Hydrolysis Of ◽  
Mild Acid

Incorporation of L-[3H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [3H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [3H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [3H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [3H]fucose, gel profiles of particulate fractions labeled with [14C]valine were much more complex.

A novel metalloproteinase present in freshly isolated rat glomeruli

1991 ◽  
Vol 260 (4) ◽  
pp. F555-F561 ◽  
Author(s):  
Q. Le ◽  
S. Shah ◽  
H. Nguyen ◽  
S. Cortez ◽  
W. Baricos
Keyword(s):  
Gel Electrophoresis ◽  
Mesangial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Significant Inhibition ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Cysteine Proteinases ◽  
Ph Optimum ◽  
Major Band ◽  
Isolated Rat

We have utilized [3H] gelatin to document high activity of a metalloproteinase present in freshly isolated rat glomeruli. [3H] gelatin degradation by glomeruli was markedly inhibited by EDTA (10 mM: -89 +/- 2.3%) and o-phenanthroline (2 mM: -72 +/- 0.1%), inhibitors of metalloproteinases. No significant inhibition of [3H]gelatin degradation was observed with inhibitors of serine or cysteine proteinases. Most (greater than 80%) of the glomerular metalloproteinase (GLOMP) activity was associated with the pellet after centrifugation of sonicated glomeruli at 100,000 g for 90 min. The pH optimum for gelatin degradation by sonicated glomeruli was approximately pH 8.5. Sodium dodecyl sulfate substrate (gelatin)-polyacrylamide gel electrophoresis revealed a single major band of EDTA-inhibitable gelatin-degrading activity with a molecular mass of approximately 116-125 kDa. The GLOMP activity was not inhibited by tissue inhibitors of metalloproteinases, did not appear to be latent, and was not activated by organomercurial activators of several latent metalloproteinases. GLOMP activity was increased 3.4-fold after incubation with trypsin (20 micrograms/ml, 25 min, 22 degrees C). These data indicate that GLOMP is distinct from the previously described matrix metalloproteinases, as well as other metalloproteinases present in the kidney, including the gelatinase secreted by cultured mesangial cells, Meprin, and endopeptidase 24.11 (enkephalinase, EC 3.4.24.11).

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