Electrophoresis acid phosphatases

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

Fia. 14. Polyacrylamide gel electrophoresis at various purification steps. Gels 1 to 4 were stained for acid phosphatase activity gel 5 was stained for protein. A current of 4 mA/gel was applied to gels 1-3 for 2 hr and to gels 4 and 5 for 6 hr. Gel 1, homogenate 2, DEAE-cellulose peak II 3, DEAE-cellulose peak I 4 and 5, crystalline enzyme. From Igarashi and Hollander (4). 

Acid phosphatase of S. aureus PS 55 is eluted from the cell surface by 1.0 M KC1 at pH 8.5. Gel filtration of this material gave a 44-fold purification. The protein seems homogeneous by gel filtration, starch block electrophoresis, and analytical ultracentrifugation with the weight of approximately 58,000 (12a). 

Another isoenzyme with substantial interest is erythrocytic acid phosphatase (EAP) (8, 9, 10). This system has three autosomal allelic genes termed A, B and C. These can be homozygous or heterozygous giving rise to BA, CA and CB phenotypes. Each of these phenotypes is easily distinguished using starch gel electrophoresis with very useful population frequencies of approximately A - 13%, B - 35%, C - 0.2%, BA - 43%, CA - 3%,... 

Figure 2. Erythrocytic acid phosphatase schematic. A schematic drawing illustrating typical results of an EAP determination in a 13%, 1mm starch gel prepared in 0.24M Nall TO, 0.15M trisodium citrate tannic acid buffer diluted 1 100. The electrophoresis is carried out for 41/2 hr at approximately 410 V. The gels are stained by the fluorescence produced after enzymatic hydrolysis by methylumbellifertjl phosphate at 37° C for 1V2 hr.

Human erythrocyte acid phosphatase (EAP) polymorphism was first described by Hopkinson, Spencer and Harris (1). EAP can be classified by electrophoresis into six different phenotypes,... 

Forensic biochemists perform blood typing and enzyme tests on body fluids in cases involving assault, and also in paternity cases. Even tiny samples of blood, saliva, or semen may be separated by electrophoresis and subjected to enzymatic analysis. In the case of rape, traces of semen found on clothing or on the person become important evidence the composition of semen varies from person to person. Some individuals excrete enzymes such as acid phosphatase and other proteins that are seldom found outside seminal fluid, and these chemical substances are characteristic of their semen samples. The presence of semen may be shown by the microscopic analysis for the presence of spermatozoa or by a positive test for prostate specific antigen. 

These results were confirmed to a large extent by Lundin and Allison (L14, L15), who examined the electrophoretic patterns of acid phosphatase from different organs and animal forms. We shall concern ourselves only with the results on human tissues. Since there is no statement that equal activities of acid phosphatase from different tissues were placed at the origin, it is difBcult to make any definite conclusions about the patterns from the different tissues. In general, these tissues showed between 10 and 17 bands upon electrophoresis at pH 6.0 for a period of about 4 hours. Human prostate had a strong band that moved very little from the origin, and this band was not seen in the other tissues. 

The acid phosphatase activity of leukocytes was studied by Valentine and Beck (B8, VI) in 1951. There appear, however, to have been no significant attempts to purify the enzyme from this source, or to describe its characteristics. Recently, Szajd and Pajdak (S32) indicated the isoenzyme characteristics of leukocyte acid phosphatase, and Li and his associates (L7, L8) studied this problem in greater detail. They suspended a leukocyte preparation, carefully separated from blood, in 5% Triton X-100 to yield a final concentration of 10 X 10 cells per milliliter and subjected the suspension to six cycles of alternate freeze-thaw treatment. The suspension was then centrifuged at lOOOp for 15 minutes at 4°C, and the supernatant was used for electrophoretic studies. Specimens centrifuged at 100,000p for 15 minutes gave the same results. Electrophoresis was carried out at 4°C for 60 minutes on a 7.5% acrylamide gel matrix containing 0.5% Triton X-100 at pH 4.0 with a current of 4 mA per tube. The substrate was -naphthyl phosphate. 

We have already discussed the properties of human erythrocytic acid phosphatase (Section 3.3), and we pointed out that, like acid phosphate in other tissues, it may exist in several isoenzymatic forms. In 1963, Hopkinson et al. (H13) subjected hemolysates of human red cells from an English population to horizontal starch-gel electrophoresis for 17 hours at 5°C. The gels were then sliced horizontally, covered with 0.05 M phenolphthalein sodium diphosphate at pH 6.0, and allowed to incubate for 3 hours at 37°C. Five different electrophoretic patterns of acid phosphatase activity could be distinguished in different individuals. Shortly thereafter Lai and his associates (L2) confirmed these findings and discovered an additional sixth pattern which had been predicted by Hopkinson et al. (H13). The distribution of these patterns in various types of population was assiduously pursued within the next several years, and several new ones were discovered in Negro populations (G3, K2). 

Differences in the electrophoretic patterns and activities between the phenotypes raised the possibility that they might also be characterized in a more specific and detailed biochemical manner. In 1949, Abul-Fadl and King (A4) observed that 0.5% formaldehyde inhibited erythrocyte acid phosphatase completely, whereas 0.01 Af l-( + )-tartrate had no effect. When 0.5% formaldehyde was added to the reaction mixture in gel electrophoresis (H13), it completely inhibited all variants of erythrocyte acid phosphatase, so that no zones of activity in any of the five types were visible after the 3-hour incubation. 

Two distinct peaks of acid phosphatase activity were detected in each phenotype, but the positions of these peaks differed. For example, in phenotype A, the peaks were approximately at tubes 150 and 190 in B, at about 130, 170, and 265 in BA, at 110 and 155. In these three, the first peaks showed minor enzyme activity. In CA, there was a major peak at about tube 130 and a smaller one at about tube 170. The shape of the curves varied according to the phenotype tested. In general, these results confirmed what gel electrophoresis originally showed, namely, that there are charge differences between the various isoenzymes. The electrophoretic patterns may also be influenced by the type of buffer used to make up the starch gel (K2). 

L14. Lundin, L. G., and Allison, A. C., Acid phosphatases from different organs and animal forms compared by starch-gel electrophoresis. Ada Chem. Scand. 20, 2579-2592 (1966). 

E9. Estborn, B., Localization of prostatic serum acid phosphatase by starch-gel electrophoresis. Clin. Chim. Acta 6, 22-24 (1961). 

A search for lysosomal hydrolases and related enzymes has been made in haemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for a-D-mannosidase, neutral o-D-glucosidase, and -D-2-acetamido-2-deoxyhexosidase. a-D-Mannosidase activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5-6.0. Electrophoresis on cellulose acetate showed three bands. Haemolysates from four patients with mannosidosis were not deficient in a-D-mannosidase. Curves of pH activity and electrophoretic patterns were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell a-D-mannosidase differs from the lysosomal acid a-D-mannosidase. 

The extents of sialylation of eleven plasma hydrolases have been examined. Most plasma hydrolases e.g. a-L-fucosidase and a-D-mannosidase) were eluted from DEAE-cellulose at a lower salt concentration after treatment with neuraminidase, although the elution profiles of p-D-glucosidase, P-D-xylosidase, and acid phosphatases were unaffected, indicating that they are less susceptible to the action of neuraminidase. The structure (9) assigned (G. Spick, B. Bayard, B. Fournet, and G. Strecker, F.E.B.S. Letters, 1975, 50, 296) to the carbohydrate component of human serotransferrin has been confirmed by high-resolution H n.m.r. spectroscopy. Electrophoresis separated human serotransferrin into four molecular forms that contain different proportions of bound iron. The nature of the interaction of renins from human and rabbit kidneys with immobilized concanavalin A, and their subsequent desorption with methyl a-D-mannopyrano-side, suggested that both proteins are glycosylated. ... 

Viewing the fact that only a portion (but not all of the acid phosphatase) of the lysosomal fraction is readily released upon physical disruption of the lysosomal membrane by freezing and thawing or by hypoos-motic pressure, Baccino et al. (1971) suggested that at least two varieties of acid phosphatase were associated with the lysosomal fraction, the first being readily, and the other not readily, dissociable from lysosomal structures. This interpretation coincides with the earlier observation of Sloat and Allen (1969) who showed two varieties of acid phosphatase associated with lysosomal fractions of rat liver. One form is readily released after physical disruption of lysosomal fractions, the other form is associated with the lysosomal membrane and became soluble only with 5% Triton X—100 treatment. This membrane-associated enzyme accounted for 40% of the total lysosomal acid phosphatase, is heat-stable, and can be separated from the soluble form by electrophoresis. But these two enzymes have similar pH optima and a common response to inhibitions by L-tartrate, fluoride, alloxan, and formaldehyde. 

The Triton X-100 solubilized microsomal acid phosphatase and the free lysosomal acid phosphatase also differ in elecfrophoretic migration rates with the lysosomal enzyme migrating faster in the polyacrylamide gel electrophoresis at an acid pH. These two enzymes can also be separated by DEAE-cellulose chromatography (Lin and Fishman, 1972). 

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