scholarly journals Partial purification and properties of the common inherited forms of adenosine deaminase from human erythrocytes

1973 ◽  
Vol 133 (1) ◽  
pp. 117-123 ◽  
Author(s):  
W. R. A. Osborne ◽  
N. Spencer
Keyword(s):  
Ionic Strength ◽  
Adenosine Deaminase ◽  
Reaction Rates ◽  
Competitive Inhibitor ◽  
Human Erythrocytes ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Gel Chromatography

1. The partial purification of adenosine deaminase, types 1, 2 and 2–1, from human erythrocytes is described. 2. The isoenzyme components characteristic of the three forms of the enzyme were partially resolved by chromatography on DEAE-Sephadex. 3. Gel chromatography of the various forms of the enzyme gave estimates of the molecular weights in the range 30000–35000. 4. Electrophoresis in starch gels containing increasing percentages of starch did not reveal any differences in molecular weight between the genetic variants or their isoenzyme components. 5. Analytical isoelectric-focusing experiments in polyacrylamide gels gave the following pI values for the four isoenzyme components present in type 2–1 erythrocytes: 4.70, 4.83, 4.94 and 5.06. 6. All forms of the enzyme gave Km values for adenosine of about 30μm and Ki values of about 8μm for the competitive inhibitor purine riboside. 7. Reaction rates of the type 1 and 2 enzymes were measured at different temperatures. Both enzymes gave values for the energy of activation for hydrolysis of adenosine of about 33.4kJ/mol (8kcal/mol). 8. Heat inactivation of all forms of the enzyme was markedly dependent on ionic strength, the rate of inactivation increasing with increasing ionic strength. The type 1 and type 2 forms of the enzyme differed significantly in their susceptibility to heat inactivation. From the variation of rates of inactivation with temperature, values were obtained for the energies of activation for the heat inactivation of both enzymes as follows: type 1 enzyme 275.5kJ/mol (65.9kcal/mol) and type 2 enzyme 241.6kJ/mol (57.8kcal/mol.).

scholarly journals Partial purification and properties of the two common inherited forms of human erythrocyte adenylate kinase

1972 ◽  
Vol 130 (3) ◽  
pp. 797-803 ◽  
Author(s):  
C. Brownson ◽  
N. Spencer
Keyword(s):  
Adenylate Kinase ◽  
Effect Of Temperature ◽  
Partial Purification ◽  
Heat Denaturation ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Increasing Temperature

1. The partial purification of adenylate kinase, types 1 and 2, from human erythrocytes is described. 2. Gel chromatography of both forms of the enzyme gave estimates of the molecular weights in the range 20000–23000. 3. Studies on crude haemolysates at various pH values indicated that the type 2 enzyme was less stable than the type 1. Heat denaturation studies on the partially purified enzymes confirmed these findings. 4. Measurements of rates of inhibition by iodoacetate and iodoacetamide showed that the type 2 enzyme reacts more readily than the type 1 enzyme with both reagents. 5. The effect of temperature on the initial velocity of ADP formation was measured at a single concentration of both AMP and MgATP2-. The two forms of the enzyme responded differently to increasing temperature.

scholarly journals Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes.

1988 ◽  
Vol 168 (1) ◽  
pp. 107-126 ◽  
Author(s):  
R E Mandrell ◽  
J M Griffiss ◽  
B A Macher
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Incubation Temperature ◽  
Human Erythrocytes ◽  
Human Infant ◽  
Blood Group Antigens ◽  
Sequence Specificity ◽  
Adult Humans ◽  
Branched Chain

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.

The Partial Purification of Two β-N-Acetyl-D-hexosaminidases from Porcine Kidney

1972 ◽  
Vol 50 (5) ◽  
pp. 563-573 ◽  
Author(s):  
Stephen J. Wetmore ◽  
Jacob A. Verpoorte
Keyword(s):  
Amino Acid ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Gel Chromatography ◽  
Porcine Kidney ◽  
Pig Kidney ◽  
Two Peaks

Two distinct fractions showing both β-N-acetyl-D-glucosaminidase (EC 3.2.1.30) and β-N-acetyl-D-galactosaminidase activity were isolated and purified from pig kidney. These preparations, which were designated A and B, were not stable during gel chromatography or prolonged dialysis. Final purifications of 600-fold for enzyme A and 440-fold for enzyme B were obtained.Gel electrophoresis and ultracentrifugation studies indicated heterogeneity in both preparations. The amino acid compositions of both preparations were very similar. Ultracentrifugation studies suggested the formation of subunits in the presence of 5 M guanidine–HCl and 1 mM dithiothreitol.A study of the enzymatic properties also showed great similarities between the two enzyme forms. Both enzymes had identical Michaelis–Menten constants of 1.88 mM for p-nitrophenyl-β-N-acetyl-D-glucosaminide and 0.38 mM for p-nitrophenyl-β-N-acetyl-D-galactosaminide. Although bovine serum albumin enhanced the activity of the enzymes it did not change the Km values. The pH-rate profiles of both enzymes with the substrate p-nitrophenyl-β-N-acetyl-D-glucosaminide showed two peaks. When p-nitrophenyl-β-N-acetyl-D-galactosaminide was used as substrate, only one peak was observed in the pH–rate profiles. However, in this case a distinct shoulder could be detected in these peaks. Heating at 50° destroyed the activities of both forms of the enzyme rapidly, but addition of bovine serum albumin protected against heat inactivation.

Dutasteride: A Review of its Use in the Management of Prostate Disorders

2011 ◽  
Vol 3 ◽  
pp. CMT.S1956
Author(s):  
Garrett D. Pohlman ◽  
Emily A. Pohlman ◽  
E. David Crawford
Keyword(s):  
Competitive Inhibitor ◽  
Open Label ◽  
Double Blind ◽  
Symptomatic Benign Prostatic Hyperplasia ◽  
Chemopreventive Agent ◽  
Adrenergic Antagonist ◽  
Urinary Tract Symptoms ◽  
Lower Urinary

Dutasteride, a synthetic 4-azasteroid, is a selective and competitive inhibitor of both type 1 and type 2 5-alpha-reductase isoenzymes approved for the treatment of men with symptomatic benign prostatic hyperplasia (BPH) who have an enlarged prostate. It has been demonstrated to be effective as monotherapy, or in combination with the alpha-adrenergic antagonist (α-blocker), tamsulosin, in several randomized, double-blind, placebo controlled trials and their open-label extensions. Treatment with dutasteride is generally safe and well tolerated. Dutasteride has become established in the management of men with lower urinary tract symptoms (LUTS) and reduces the risk of acute urinary retention and surgery related to BPH. Dutasteride may also have a role as a chemopreventive agent in the future as emerging evidence demonstrates a reduced risk of prostate cancer with this agent.

Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes

1999 ◽  
Vol 277 (5) ◽  
pp. C926-C936 ◽  
Author(s):  
Isabel Bize ◽  
Birol Güvenç ◽  
Aeisha Robb ◽  
Guido Buchbinder ◽  
Carlo Brugnara
Keyword(s):  
Ionic Strength ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Human Erythrocytes ◽  
Phosphatase Inhibitors ◽  
Pp2a Activity ◽  
Highly Sensitive ◽  
Protein Phosphatase Inhibitors ◽  
Membrane Bound

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 ± 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 ± 10% per 100 meq/l). Increases in membrane PP2A activity (270 ± 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 ± 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.

Type 2 Diabetes Overtakes Type 1 in Hispanic Girls

2008 ◽  
Vol 38 (15) ◽  
pp. 18
Author(s):  
SHERRY BOSCHERT
Keyword(s):  
Type 2 Diabetes
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