6-Phosphofructo-1-kinase isoenzymes of the jejunal mucosa of rabbit, rat and mouse (original) (raw)
Related papers
Journal of Biological Chemistry, 1980
Isozymes (PGK-1 and PGK-2) and genetic variants (PGK-2A, PGK-2B, and PGK-2C) of 3-phosphoglycerate kinase were purified by affinity chromatography using an 8-(6-aminohexyl)-amino-ATP-Sepharose column as the key step. Antisera raised against purified PGK-1 and PGK-2A were tested for specificity and cross-reactivity by application of double immunodiffusion and enzyme immunoinactivation methods. By double immunodiffusion, no precipitin lines were observed between anti-PGK-2A and PGK-1, but a weak cross-reactivity between anti-PGK-1 and PGK-2A was detected. In addition to specific inhibition of PGK-1 and PGK-2A by their respective antisera, anti-PGK-1 was shown to inhibit PGK-2 activity at high antiserum concentrations, whereas no inhibition of PGK-1 activity by anti-PGK-2A was observed. The amino acid compositions of PGK-1 and PGK-2 revealed a certain degree of homology. However, tryptic peptide maps showed no obvious similarity in the peptide spots between these two 3-phosphoglycerate...
Altered phosphoglycerate kinase from old rat muscle shows no change in primary structure
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1985
Phosphoglycerate kinase (ATP:3-phospho-D-glycerate l-phosphotransferase, EC 2.7.2.3) from young and old rat muscle was purified to homogeneity. After ascertaining that each preparation of the enzyme obtained from the latter indeed possessed altered properties, matched pairs of young and old enzymes were subjected to amino acid analysis and peptide mapping by HPLC. Following S-carboxymethylation, the respective young and old enzymes were digested with each of the following three proteinases: trypsin, chymotrypsin and S. aureus V8 proteinase. The corresponding peptides were resolved by reverse-phase HPLC. The peptide patterns obtained from both enzyme forms were identical. Even when the peptides obtained from digestion of phosphoglycerate kinase with S. aureus V8 proteinase were further digested with trypsin, no differences were observed. Comparative amino acid analyses also showed no differences. These results provide direct evidence that there are no changes in the sequence of altered rat muscle phosphoglycerate kinase and support the hypothesis that the differences in properties between the young and old forms of the enzyme result from a conformationai modification.
Distribution and properties of rat intestinal alkaline phosphatase isoenzymes
Experimental animals, 2001
The ALP activities and properties of rat intestine cut into 20 segments were examined, and we were able to demonstrate that the ALP activity of upper intestine is high compared to that of lower intestine. This result coincided with those of other reports. However, we newly clarified that there is an ALP isoenzyme found in the lower intestine which can be inhibited by L-homoarginine. The molecular weight of the ALP isoenzyme was 136 kDa. In addition, it was clarified that there are several isoenzymes from upper to lower intestine. This study demonstrates that there exist isoenzymes, which are inhibited by L-HArg, in the intestine which are similar to the isoenzymes in the liver, bone and kidney.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1981
In the small bowel mucosa of the rot, alkaline phosphatase was found to be present in the 105 000 × g supernatant fraction, in addition to the major brush border membrane-bound form. This soluble enzyme contributed up to 3.6% of total alkaline phosphatase activity in the adult rat. Combined use of inhibitors of serine, thiol and carboxyl proteases during homogenization did not significantly affect the proportion of enzyme activity in the supernatant fraction. In suckling rats a much larger proportion of alkaline phosphatase was soluble, reaching 36.5% in the 14-day-old animal. The soluble and membranous forms were compared in adult and suckling rats by examining their biochemical and immunological characteristics. In the adult rat the specific activity of membranebound alkaline phosphatase showed a progressive fall distal to the duodenum, while maximal specific activity of the supernatant enzyme occurred in the distal ileum. By contrast, in the suckling rat the specific activity of both forms increased from the duodenum to the ileum. Soluble alkaline phosphatases in mature and immature rats were distinct from the membranous forms when examined by polyacrylamide gel electrophoresis in the presence of Triton X-100. Electrophoretic heterogeneity was seen in the soluble adult enzyme, characteristic Rf values occurring for each level of the small bowel (i.e., duodenum, jejunum and ileum). Such heterogeneity was not seen in suckling rats. Antigenically, the soluble and membranous forms were identical to each other in adult and suckling rats. Analysis of mucosal cells isolated sequentially from tip of the villus to crypt showed that both the soluble and membranous isozymes had highest activity towards the tip of the villus. We conclude that the soluble and membranous forms are distinct biochemically and yet are antigenically identical and arise from the same level of the villus. The presence of the soluble form, which is not an artifact of tissue preparation, indicates that alkaline phosphatase is not exclusively an integral membrane protein.
Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine
European Journal of Biochemistry, 1993
A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acidmol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components.
Differences between duodenal and jejunal rat alkaline phosphatase
Clinical Biochemistry, 2000
The aim of this study was to kinetically characterize rat tissue-nonspecific-alkaline phosphatase (TNS-ALP) and intestinal (duodenal-and jejunal-IALP), and to determine the effect of substances known to affect phosphorylation/dephosphorylation on TNS-and IALP activity. Design and results: The ranking order of ALP activity (K enzyme ) was duodenal mucosa (IALP) Ͼ jejunal mucosa (IALP) Ͼ kidney (TNS-ALP) Ͼ brain (TNS-ALP). Levamisole was found to produce a concentration-dependent decrease of ALP activity in kidney and brain. However, levamisole had no effect on duodenal ALP activity and produced a concentrationdependent increase on jejunum ALP activity. In brain and jejunum homogenates, octreotide, a stable somatostatin analogue, produced a concentration-dependent increase in ALP activity. In relation to duodenum ALP activity, octreotide produced a biphasic effect. Reverse transcription-polymerase chain reaction showed the presence of IALP-I mRNA both in duodenal and jejunal mucosa, but IALP-II only in duodenal mucosa.
Biochimica et Biophysica Acta (BBA) - Enzymology, 1981
The posslbdlty of a funchonal complex formahon between glyceraldehyde-3-phosphate dehydrogenase (EC 1 2 1 12) and 3-phosphoglycerate kmase (EC 2 7 2 3), enzymes catalysmg two consecuhve reactions m glycolysls has been investigated Kinetic analysis of the coupled enzymatic reaction dul not reveal any kmehc sign of the assumed interaction up to 4 10-6 M kmase and 10-4 M dehydrogenase Fluorescence amsotropy of 10-7 M or 2 10-s M glyceraldehyde-3-phosphate dehydrogenase labeled with fluoresceln isotMocynate did not change in the presence of non-labeled 3-phosphoglycerate kmase (up to 4 10-s M) The frontal gel chromatographic analysis of a mixture of the two enzymes (10-4 M dehydrogenase and I0-s M kmase) could not reveal any molecular species with the kinase activity hawng a molecular weight higher than that of 3-phosphoglycerate kmase Both types of physlcochemlcal measurements were also performed m the presence of substrates of the kmase and gave the same results The data seem to mvahdate the hypothesis that there is a complex between purified pig muscle glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kmase
Molecular properties of rat intestinal alkaline phosphatase
Biochim Biophys Acta Protein, 1976
Rat intestinal alkaline phosphatase is an heterogeneous glycoprotein that contains three protein sub-forms separable by electrophoresis. The molecular weight for the glycoprotein (i.e. the average for the three sub-forms) is 157 000-160 000. Three protein sub-forms are detectable on sodium dodecyl sulphate-polyacrylamide gel electrophoresis that migrate at rates corresponding with molecular weights of 64 000, 79 000 and 92 000. Treatment of native alkaline phosphatase with 6 M guanidine. HCI or buffer at pH 3.0 results in a product with a molecular weight of 78 000 and 70 000, respectively. Thus it is concluded that each of the three sub-forms is a dimer of identical or closely similar subunits. Limited proteolysis results in the production of new enzymically active sub-forms separable by electrophoresis. Using a bacterial protease it is possible to convect intestinal alkaline phosphatase into a form with a molecular weight of 132 000 without causing any significant change in kinetic properties. Electrophoresis of this new form on sodium dodecyl sulphate polyacrylamide gel suggests that it is composed of 66 000-dalton subunits. The native enzyme contains at least 20 ~ by weight of carbohydrate that probably contributes to microheterogeneity of a second degree superimposed on that stemming from the presence of three protein sub-forms. Treatment with various glycosidases has no effect on electrophoretic behaviour, however. It is suggested that the three sub-forms possibly represent different stages of a maturation process that operates by limited proteolysis of a single parent protein.
Journal of Biological Chemistry
In the suckling rat intestine, alkaline phosphatase is present in both soluble and membranous forms. These forms were purified from the 105,000 X g ileal supernatant and jejunal particulate fractions, respectively. Protease inhibitors added to the homogenate buffer did not significantly alter the yield of soluble and membranous enzyme. Moreover, cytoplasmic fractions from suckling rat ileum containing active lysosomal enzymes degraded rather than solubilized the membranous form. The two forms were found to be kinetically similar and immunologically identical. The soluble form had M, = 108,000 in nondenaturing sodium dodecyl sulfate gels and a single subunit in denaturing sodium dodecyl sulfate gels with M, = 47,000. Comparable values for the membranous forms were M, = 130,000 and 66,000. With Triton polyacrylamide gel electrophoresis, the RF of the soluble form was 0.48, faster than the membranous form (RF 0.20). These values were identical for crude and purified enzymes. Analysis of carbohydrate content revealed that the soluble form contained more fucose (20 versus 8 mol of sugar/g of protein X lo-') and N-acetylgalactosamine (32 versus 14 mol of sugar/g of protein X lo-') than did the membranous form. The membranous enzyme displayed strong lipid affinity by ready incorporation into liposomes, while the soluble form failed to be included. The two enzyme forms had different patterns of molecular heterogeneity by isoelectric focusing, with bands of activity at pH 4.97 and 5.06 for the soluble form and 4.33, 4.43, and 4.60 for the membranous enzyme. The papainsolubilized membranous enzyme migrated similarly to the soluble form on Triton-and sodium dodecyl sulfate gel-electrophoresis but reproduced the pattern of the membranous form on isoelectric focusing. From these studies, we conclude that: (a) the soluble and membranous forms of intestinal alkaline phosphatase are closely related but distinct isozymes; and (b) the soluble form is not simply a proteolytically solubilized product of the membranous enzyme.