Expression pattern of 5''-nucleotidase in Dictyostelium.

In order to analyze the expression pattern of the 5′-nucleotidase (5nt) gene in Dictyostelium, we made a fusion construct in which the 5nt promoter directed the expression of β-galactosidase gene. The reporter gene was not active in vegetative amoebae but was expressed during the aggregation stage. At the slug stage, 5nt was highly expressed in pstAB cells. As the slug moved along the substratum, high activity of β-galactosidase was detected in cells that were left behind in the slime trail. In the completed fruiting body, 5nt was expressed in the lower cup, the anterior like cells (ALC) and the basal disc.     

The molecular forms of gamma-glutamyl transferase in bile and serum of icteric rats

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.     

Structural differences between plasma-membrane 5''-nucleotidase in different cell types as evidenced by antibodies.

Antibodies raised against bovine 5'-nucleotidase inhibit this enzyme as well as 5'-nucleotidase from other bovine tissues, showing common structure(s) between these proteins. However, an IgG fraction directed against the glucidic moiety of the liver enzyme did not cross-react with the enzyme from lymphocyte or caudate nuclei, a clear indication that within the same species the 5'-nucleotidase differs from one cell type to another. In addition, immunoblots after electrophoresis show that the previous antibodies recognize 5'-nucleotidase from human, mouse or chicken origin. However, only human 5'-nucleotidase activity can be inhibited by the antibodies. Thus at least three groups of antigenic determinants must exist on the 5'-nucleotidase: one related to the glucidic moiety of the glycoprotein whose binding inhibits the enzyme activity, another related to the catalytic site, as its binding also led to enzyme inhibition, and a last one of structural nature. It seems that the third group of determinant is common to many species, whereas the second one is more restricted.     

Purification, characterization and cellular localization of 5''-nucleotidase from Torpedo electric organ.

5'-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5'-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.     

Subcellular distribution and movement of 5''-nucleotidase in rat cells.

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.     

Partial purification and properties of an AMP-specific soluble 5''-nucleotidase from pigeon heart.

A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.     

Multiple forms of gamma-glutamyltransferase in normal human liver, bile and serum.

A study of the multiple forms of gamma-glutamyltransferase ((gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2) in normal human liver, bile and serum are reported. An amphiphilic form of the enzyme was demonstrated in all three samples. When solubilized with detergent, estimated values for Stoke's radius of 48 A and sedimentation coefficient of 5 S were obtained for this form. A hydrophilic form was also present in serum and bile, which showed identical properties to the enzyme form obtained after papain-treatment of the three samples. The Stoke's radius was found to be 37 A, and the sedimentation coefficient 5 S. It was concluded that the heterogeneity of enzyme activity found both on gel filtration and on electrophoresis was due to aggregates of the amphiphilic form with lipids and other proteins, and could not be ascribed to the presence of isoenzymes.     

Recycling of 5''-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.     

Studies on blood serum beta-N-acetylglucosaminidases from several mammalian species--separation of different enzyme forms.

1. The beta-N-acetylglucosaminidases studied from peripheral blood sera have the following activities: 55.6 mU/ml in Bos taurus L. (bull, "Morucha" race), 31.3 mU/ml in Sus scropha var. domestica L. (pig) and 3.7 mU/ml in Capra hircus L. (goat). 2. The pH optima of the enzymes are 4.5 for Bos taurus L. and Sus scropha L., and 5.0 for Capra hircus L. 3. Two enzyme forms, A and B, have been separated from Bos taurus L. and Capra hircus L. sera by DEAE-cellulose chromatography; and three forms, A, B and S, from Sus scropha L. serum. 4. The B forms are more heat-stable than the A and the S forms. The S form has intermediate heat-stability between the A and the B forms.     

Acetylcholinesterase molecular forms in serum and erythrocytes of laboratory animals

The distribution of the molecular forms of acetylcholinesterase in the blood of various animal species was examined. The globular tetrameric form was most frequently observed in serum but mouse serum also contained a globular monomer. Globular monomers (rat) dimers (mouse, dog, rabbit) and tetramers (dog) were found in erythrocytes. Interspecies differences make it difficult to formulate a cohesive theory as to the origin and function of blood-borne enzymes.     

Acetyl- and butyrylcholinesterase molecular forms in normal and streptozotocin-diabetic rat retinal pigment epithelium.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinal pigment epithelium (RPE). Tissues were sequentially extracted with saline (S(1)) and saline-detergent buffers (S(2)). About a 50% decrease in AChE molecular forms was observed in the diabetic RPE compared to the controls. Approximately 70% of the BChE activity in normal RPE was brought into solution and evenly distributed in S(1) and S(2). Analysis of the fractions from RPE revealed the presence of G(A)(1), G(A)(4) and a small proportion of G(H)(4) BChE forms in S(1); whereas G(A)(4) and G(A)(1) molecules predominate in S(2). A 40% decrease in the activity of G(A)(4) in S(2) was observed in the diabetic RPE. Our results show that diabetes caused a remarkable decrease in the activity of cholinesterases molecular forms in the RPE. This might be related to the alterations observed in diabetic retinopathy.     

Gangliosides in blood serum of normal rats and Morris hepatoma 5123tc-bearing rats.

The effects of malignancy upon blood serum ganglioside patterns were investigated. Lipids extracted from the blood serum of Morris hepatoma 5123tc-bearing rats were characterized by severalfold increases in the content of hematosides, monosialogangliosides and disialogangliosides, as compared with lipids extracted from the serum of normal rats. However, the content of trisialogangliosides in lipids extracted from the serum of cancer-bearing rats substantially decreased. In general, the change in the profile of gangliosides in blood serum reflects, but is less pronounced, than that observed in the comparison of Morris hepatoma tissue to normal liver tissue.