scholarly journals The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine

1983 ◽  
Vol 211 (2) ◽  
pp. 373-379 ◽  
Author(s):  
S M Khoja ◽  
N L Beach ◽  
G L Kellett
Keyword(s):  
Small Intestine ◽  
Specific Activity ◽  
Proteinase Inhibitors ◽  
Deae Cellulose ◽  
Native Enzyme ◽  
Rat Small Intestine ◽  
Cellulose Acetate Electrophoresis ◽  
Isolation And Characterization ◽  
Main Component ◽  
Proteolytic Product

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.

scholarly journals Phosphofructokinase D from the epithelial cells of rat small intestine

1983 ◽  
Vol 215 (2) ◽  
pp. 335-341 ◽  
Author(s):  
S M Khoja ◽  
G L Kellett
Keyword(s):  
Skeletal Muscle ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Cellulose Acetate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Intestinal Epithelial ◽  
Rat Small Intestine ◽  
Cellulose Acetate Electrophoresis ◽  
Type C

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

Intestinal phospholipase A and triglyceride lipase: localization and effect of fasting

1982 ◽  
Vol 242 (2) ◽  
pp. G168-G176 ◽  
Author(s):  
K. M. Shakir ◽  
L. Gabriel ◽  
S. G. Sundaram ◽  
S. Margolis
Keyword(s):  
Small Intestine ◽  
Specific Activity ◽  
Isolated Hepatocytes ◽  
Phospholipase A ◽  
Intestinal Cells ◽  
Enzyme Release ◽  
Rat Small Intestine ◽  
Triglyceride Lipase ◽  
Plasma Hormones ◽  
Fasted Rats

We investigated the distribution of phospholipase A and triglyceride lipase in the rat small intestine and the effects of heparin and hormones on enzyme release. Phospholipase A activity was 10 times higher in the ileum than in the jejunum; triglyceride lipase activity was threefold higher in the jejunum than in the ileum. Activities of both enzymes were much greater in villus than in crypt cells. The specific activity of phospholipase A was highest in microsomes and least in cytosol. The crude nuclei and brush-border fraction contained 40.5% of total phospholipase A activity; mitochondria contained 33.8%; and microsomes, 17.4%. Phospholipase A activity increased significantly in the distal intestinal mucosa in fasted rats compared with controls. Heparin did not increase the release of phospholipase A by isolated intestinal cells or perfused intestinal vasculature. Thus, the small intestine probably does not contribute significantly to the phospholipase A activity of postheparin plasma. Hormones and cAMP, which inhibit the secretion of phospholipase A and triglyceride lipase from isolated hepatocytes, had no effect on the release of either enzyme from intestinal cells.

scholarly journals Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

1983 ◽  
Vol 209 (2) ◽  
pp. 497-503 ◽  
Author(s):  
N Uldbjerg ◽  
A Malmström ◽  
G Ekman ◽  
J Sheehan ◽  
U Ulmsten ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Uterine Cervix ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Average Molecular Weight ◽  
Deae Cellulose ◽  
Sedimentation Equilibrium ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Isolation And Characterization

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

Isolation and Characterization of a Pancreatic Elastase from Plasma of Patients with Acute Pancreatitis

1982 ◽  
Vol 62 (3) ◽  
pp. 321-328 ◽  
Author(s):  
Naotika Toki ◽  
Sumiyoshi Takasugi ◽  
Hiroyuki Sumi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Isolation And Characterization ◽  
Bean Trypsin Inhibitor

1. An elastase-like enzyme in plasma of patients with acute pancreatitis was purified by DEAE-cellulose column chromatography and polyacrylamide-gel disc electrophoresis. 2. In this way 0.24 mg of purified enzyme with a specific activity of 3.94 succinyl-l-alanyl-l-alanyl-l-alanyl-p-nitroanilide units/mg of protein was obtained from 10 ml of plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel disc electrophoresis and had an apparent molecular weight of 24 000 as measured by gel filtration on Sephadex G-100. 4. This enzyme hydrolysed denatured casein and Congo Red—elastin as well as succinyl-l-alanyl-l-alanyl-l-alanyl-p-nitroanilide. Its amidolytic activity was inhibited by soya bean trypsin inhibitor, but not by aprotinin. 6. We propose that an elastase-like enzyme, probably different from elastase 1 or elastase 2, is liberated from the pancreas into blood during acute pancreatitis and becomes combined with α2-macroglobulin.

Neurotensin stimulates [3H]oleic acid translocation across rat small intestine

1986 ◽  
Vol 251 (6) ◽  
pp. G823-G829 ◽  
Author(s):  
M. J. Armstrong ◽  
M. C. Parker ◽  
C. F. Ferris ◽  
S. E. Leeman
Keyword(s):  
Small Intestine ◽  
Oleic Acid ◽  
Mesenteric Artery ◽  
Specific Activity ◽  
Femoral Vein ◽  
Lymph Flow ◽  
Entire Length ◽  
Intestinal Lumen ◽  
Rat Small Intestine ◽  
Series Of Experiments

The effect of neurotensin (NT) on the translocation of intraluminally administered lipid across the duodenum as well as across the entire length of the small intestine was studied in the rat. In the first series of experiments, the appearance in the lymph of [3H]oleic acid instilled as a bolus into a segment of the duodenum was followed for 3 h. Infusion of NT (0.6 pmol X kg-1 X min-1) via the superior mesenteric artery resulted in a significant increase in the appearance of label in the lymph when compared with saline infusion (17.3 +/- 3.1 vs. 8.6 +/- 1.4%, P less than 0.01, respectively). In the second series of experiments, lipid was infused into the entire length of the small intestine over 4 h, and the accumulation of label in the lymph was measured. The infusion of NT (1.0 pmol X kg-1 X min-1) into the femoral vein also significantly increased the appearance of label when compared with animals infused with saline (49.0 +/- 2.0 vs. 34.2 +/- 5.2%, P less than 0.05, respectively). In this study, the specific activity of the triglyceride recovered in the lymph was higher in the rats given NT than in the controls (P less than 0.05). No significant changes in lymph flow were observed as a consequence of NT infusion. These results indicate that NT infusion into the circulation increases the translocation of oleic acid from the intestinal lumen into the lymph of rats.

scholarly journals The effect of starvation on the control of phosphofructokinase activity in the epithelial cells of the rat small intestine

1983 ◽  
Vol 210 (1) ◽  
pp. 129-135 ◽  
Author(s):  
A Jamal ◽  
G L Kellett
Keyword(s):  
Small Intestine ◽  
Epithelial Cells ◽  
Terminal Ileum ◽  
Specific Activity ◽  
Utilization Rate ◽  
Proximal Jejunum ◽  
Rat Small Intestine ◽  
Humoral Factors ◽  
Specific Activities ◽  
Regulatory Properties

1. The effect of depriving rats of food for 48 h on the specific activity of phosphofructokinase in the epithelial cells of the small intestine and on the regulatory properties of the enzyme displayed in crude (particle-free) mucosal extracts was studied. 2. The specific activity of phosphofructokinase, measured under optimal conditions at pH8, in the mucosa of fed rats showed a negative aboral gradient along the intestine, decreasing from 15.2 +/- 1.2 units (mumol/min)/g wet wt. in the proximal jejunum to 4.6 +/- 1.2 units/g wet wt. in the terminal ileum. 3. After starvation, the gradient was diminished, but not abolished; the diminution in gradient was due almost exclusively to a decrease in the specific activity of phosphofructokinase in the proximal jejunum by about 30%, there being no change in the terminal ileum. 4. In fed rats, the susceptibility of phosphofructokinase to inhibition by ATP, when assayed in crude mucosal extracts under suboptimal conditions, was independent of length along the small intestine; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM-fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8, v0.5/V, was 0.36 +/- 0.05 in the proximal jejunum and 0.42 +/- 0.07 in the terminal ileum. 5. After starvation, the susceptibility of phosphofructokinase to inhibition by ATP was increased and was again found to be independent of length along the small intestine: after starvation, v0.5/V was 0.19 +/- 0.04 and 0.20 +/- 0.07 for the proximal jejunum and the terminal ileum respectively. 6. Re-feeding of previously starved rats on a high-carbohydrate diet overnight for 16 h restored both the specific activities of phosphofructokinase and its susceptibility to inhibition by ATP to normal values for fed rats. 7. The data support the idea that the specific activities and the regulatory properties of phosphofructokinase in the epithelial cells of rat small intestine are mediated by distinct humoral factors. 8. The changes in glucose utilization rate of the jejunum when rats are starved can in principle be accounted for by a combination of changes in the specific activity and in the regulatory properties of mucosal phosphofructokinase.

scholarly journals The effect of pectin on the structure and function of the rat small intestine

1979 ◽  
Vol 42 (3) ◽  
pp. 357-365 ◽  
Author(s):  
R. C. Brown ◽  
J. Kelleher ◽  
M. S. Losowsky
Keyword(s):  
Small Intestine ◽  
Small Bowel ◽  
Specific Activity ◽  
Basal Diet ◽  
Muscle Layer ◽  
Glucose Absorption ◽  
Structure And Function ◽  
Rat Small Intestine ◽  
And Function

1. The effect of pectin on the structure and function of the rat small intestine was compared with that of a standard pellet diet and of a fibre-free basal diet.2. The length and wet weight of the small bowel was significantly greater inpect in-fed rats than in either pellet- or basal-diet-fed rats.3. Histological measurements of longitudinal sections from the small bowel showed a significantly greater crypt depth and muscle layer thickness in the mid-jejunum and ileum of the pectin fed rats. Villous height showed less variation.4. The specific activity of alkaline phosphatase (EC 3.1.3.1)and leucyl-P-naphthylamidase (EC 3.4.11.1) in mucosal scrapings was significantly lower in the upper jejunum of pectin-fed rats compared with either of the other dietary groups. The differences were not so marked in mid-jejunum or ileum.5. Glucose absorption measured in vivo from jejunal and ileal loops was similar in all three dietary groups.6. With two minor exceptions there were no significant differences in any of these measurements between the pellet- and basal-diet-fed rats.7. These findings could be explained by increased epithelial cell turnover caused by pectin. The possible mechanisms of this are discussed.8. The effect of pectin on the human small bowel requires study before it can be widely prescribed in man.

STUDIES ON THE DISTRIBUTION AND KINETICS OF THE ALKALINE PHOSPHATASE OF RAT SMALL INTESTINE

1959 ◽  
Vol 37 (1) ◽  
pp. 699-709 ◽  
Author(s):  
Eugenie Triantaphyllopoulos ◽  
Jules Tuba
Keyword(s):  
Alkaline Phosphatase ◽  
Small Intestine ◽  
Specific Activity ◽  
Supernatant Fraction ◽  
Magnesium Ion ◽  
Rat Small Intestine ◽  
Albino Rat ◽  
Michaelis Constants ◽  
Intestinal Enzyme ◽  
Kinetics Of

Alkaline phosphatase activity was demonstrated throughout the entire length of the small intestine of the albino rat and the relative amounts present seemed to decrease exponentially from the pylorus to the ileocolic valve. All subcellular fractions of intestinal homogenates were found to hydrolyze sodium β-glycero-phosphate. The distribution of activity of the enzyme was as follows: "microsomes", about 76%; nuclei, 10%; mitochondria, 8%; and in the nongranular, supernatant fraction, 9%. The specific activity for these fractions was: 52, 3, 10, and 3, respectively. The effects of the time of incubation, pH of the hydrolytic mixture, and the concentration of the enzyme were similar in all fractions. For the enzyme in the various fractions slight differences were observed in the values of the Michaelis constants, and in the degree of activation by magnesium ion. These variations may be explained on the basis of differences in the accessibility of substrate and magnesium ion to the enzymes in the various fractions. The finding that the "microsomes" contained the highest levels of alkaline phosphatase suggests that this intestinal enzyme is produced almost entirely by these submicroscopic particles.

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