Enzymic activities from isoelectric

Figure 3. pH diagram with distribution of enzymic activities from isoelectric separation. The fraction volume was 2.3 ml. (2)... 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Albersheim and Killias 167) purified the enzyme ninefold from Pectinol R-10. The optimal pH for activity was 5.1-5.2, and its isoelectric point was between 3 and 4. Pectin lyase was active on 65% esterified citrus pectin but not on polygalacturonic acid. The enzyme was more active in citrate and phosphate buflFers than in acetate. The addition of CaCL> to reaction mixtures buflFered with citrate or phosphate inhibited the reaction. Product inhibition was markedly increased by addition of plant auxins 168). Bull 169) suggested that the auxin eflFects were artifacts in spectrophometrically dense reaction mixtures. 

Porcine pancreas and porcine pancreatic juice appear to contain a single protein endowed with lipolytic activity. As stated earlier, this protein corresponds to about 2.5 % of the total proteins of the juice. Its molecular activity (turnover number) is likely to be higher than 300,000 under the conditions of the test. Shortage of pure material has thus far prevented any investigation of its molecular properties. It is merely known to be quite soluble in water, to have an isoelectric point of 5.2 in 0.025 M acetate buffer, and to give a conventional protein spectrum. Lipase present in pancreatic juice is likely to be identical with the enzyme extracted from pancreatin. 

Internal markers or standard proteins have most often been used by researchers to assure themselves that separations by electrophoresis are consistent and reproducible (21). In addition, charge markers In Isoelectric focusing are also used, although standard preparations of these materials are less reliable, especially under denaturing conditions. Other Internal standards that are often reported are materials for assuring radlolabel activity, enzyme activity, or other materials to monitor repeatability of measurements. In all cases, the stability of the standard material Is the key to long-term quality control. Large, batches of standards that are reproducible from lot to lot would also be useful. 

Fig. 1. Denaturing isoelectric focusing of APP T. Hemolysate samples enriched for enzyme activity were focused and analyzed for immunoreactive material using the protein blot technique. Panel A normal. Lanes A and C L. family. Lane B (L.L.) H. family. Lanes D (A.S.), E (F.D.H.), F (C.D.H.), G (J.E.S.), and H (L.S.) B. family. Lanes I (N.B.) and J. (S.B.) and D. family. Lanes K (M.D.) and L (B.D.). Panel B Lane A, F.R. Lane B, S.R. Lane C, M.R. and Lane D, normal. Figures were taken from reference 4.

Immunochemical and histological investigations of tissues from the oligodendrocytes of human brain have revealed the presence of glycoproteins, and L-fucose-containing glycoproteins are present in the perikaryal fraction of neurons. Comparisons of the substrate specificity, isoelectric point, enzymic activity, and optimum pH have shown that hexosaminidase C is identical with the principal residual activity in SandhofT s fibroblasts. ... 

The D-glucose oxidase from Aspergillus niger binds concanavalin A to form an enzymically active precipitate, which is dissociated by methyl a-D-gluco-pyranoside. The adsorption, desorption, and activity of this D-glucose oxidase on selected clays followed the pattern exhibited by other proteins, with maximum adsorption onto the clay occurring at, or below, the isoelectric point. ... 

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