Hydrophobic properties of alkaline phosphatases

The butanol extraction method of Morton (1950), a routine step in enzyme purification, is discussed with special reference to a hydrophobic form of alkaline phosphatase from human liver tissue. This form slowly precipitates from butanol-extracted liver tissue homogenates stored at 4 degrees C. Furthermore, it is lost when acetone precipitation is applied as a purification procedure. The soluble form in liver tissue is shown to have a higher relative hydrophobicity than the serum liver/bone isoenzyme. The use of sodium cholate in the isolation of the hydrophobic form produces an artefact in isoelectric focusing, which can be abolished by dialysis prior to focusing.     

Isoelectric focusing studies on the neutral 5'-nucleotidase from Wistar rat hippocampus: evidence for the absence of isoenzymes.

The pattern and some substrates characteristic of the rat brain 5'-nucleotidase were studied using the isoelectric focusing technique, which revealed that the enzyme is present in a single form in hippocampus extracts. An alkaline phosphatase, which is also able to split nucleoside monophosphates, is not active at neutral pH values. The isoelectric points were found to be 6.4 +/- 0.1 for the specific 5'-nucleotidase and 6.8 +/- 0.1 for the phosphatase.     

Studies on naturally occurring proteinous inhibitor for transmethylation reactions

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.     

Detection of alkaline phosphatase activity after conventional isoelectric focusing by an indoxyl-tetrazolium salt technique

Alkaline phosphatase was solubilized from human and rat tissues using papain in the presence of TRITON X-100 and subjected to isoelectric focusing (IEF) in polyacrylamide or agarose gels. Up till now, usually 1- and 2-naphthylphosphates have been used as substrates in order to specifically stain molecular forms of this enzyme by the azo-dye technique. In this paper, the use of another histochemical substrate, 5-bromo-4-chloro-3-indoxyl phosphate, in combination with tetrazolium salts [McGadey, J. (1970) Histochemie 23, 180-184] is presented. After hydrolysis, the released indoxyl moieties reduce tetrazolium salts to insoluble formazans at the zones of alkaline phosphatase activity. Zymogrammes showing molecular forms of alkaline phosphatase from 20 rat organs and the application of this staining technique for the detection of alkaline phosphatase activity in non-dialyzed human plasma after IEF are presented.     

Placental alkaline phosphatase types and subtypes determined by agarose gel electrophoresis and separator isoelectric focusing

Summary Two techniques for phenotyping the human placental alkaline phosphatase system were developed: high-voltage agarose-gel electrophoresis and thin-layer separator isoelectric focusing on agarose. These methods enabled a more rapid and sensitive phenotyping of all common phenotypes than the traditionally employed starch-gel electrophoresis. An extended polymorphism of placental alkaline phosphatase was revealed by isoelectric focusing. The existence of two suballeles of Pl¹ allele and two suballeles of Pl² allele was postulated.     

An isoelectric focusing study of acid phosphatase heterogeneity in rat liver

The different forms of acid phosphatase (EC 3.1.3.2) in rat liver homogenates, lysosomal, mitochondrial, microsomal fractions and cytosol were studied with isoelectric focusing. Evidence is presented that isoelectric focusing of acid phosphatase in subcellular fractions shows individual changes and time related patterns. Mild autolysis shifted all enzyme activity peaks of isoelectric focusing patterns to the one at pH 7.04.     

Separation of membrane proteins in an undenatured form by isoelectric focusing

Conditions are described which allow good resolution of membrane proteins in an undenatured form by isoelectric focusing in thin polyacrylamide gels in Triton X-100. High voltages and deionization of the acrylamide are essential. Streaking is grossly reduced by sample application in Bio-Gel P-60, by deionization of the Triton, and dialysis of membrane samples against low ionic strength buffer at slightly alkaline pH. The latter step also greatly improves solubilization of the membrane components. Reproducible isoelectric focusing patterns of proteins from red cell, thyroid, and lymphocyte membrane are presented.     

Scanning isoelectric focusing of cholera enterotoxin in polyacrylamide gels

Scanning isoelectric focusing has been employed for continuous monitoring of the isoelectric spectrum of highly purified cholera enterotoxin in 4% polyacrylamide gels containing 2% ampholytes pH 3–10. The resolution obtained by this technique is of high order because at no instance during focusing interruption of current occurs and thus diffusion of the isolated protein moieties is suppressed. An added aspect of scanning isoelectric focusing was that it allowed estimation of the minimal focusing time of cholera enterotoxin. Thus under the standard assay procedure, the main basic component of cholera enterotoxin was focused in 5800 sec, while the other at least 3 minor acidic and anodic components were focused in approximately 19000 sec. Focusing of cholera enterotoxin in the presence of 6m urea allowed the visualization of 5 well defined and about equal components. The proteinaceous nature of the observed peaks was verified by scanning at wavelengths other than 280 nm, staining of gels for protein, and varying the concentration of the enterotoxin. The design of scanning isoelectric focusing equipment is presented. Reproducibility, economy of sample, and ampholytes and simplicity of experimental technique were some of the features of this apparatus. The resolution of scanning isoelectric focusing was found to be superior to that of ordinary disc and SDS gel electrophoresis.     

Combined immunoabsorption and isoelectric focusing of barley and malt amylases in polyacrylamine gel.

A technique combining immunoabsorption, isoelectric focusing, and enzymatic characterizationin the same polyacrylamide gel is described. The a- and 0-amylases from barley seeds, an immune serum induced in rabbits by barley malt α-amylase, the immunoglobulin G (IgG) of the immune serum, and the IgG purified from a nonimmunized animal were used. The application of this technique in physiological and genetical studies to the identification of amylolytic enzymes which cannot be distinguished by existing chromogenic reactions and which have similar isoelectric points is discussed.     

Determination of isoelectric points in thin-layer isoelectric focusing: The importance of attaining the steady state and the role of CO2 interference

Isoelectric points differing by 1 to 2 pH units are measured for horseradish peroxidase and lactoperoxidase depending upon the technique of isoelectric focusing, namely, the density gradient technique or systems stabilized by either granulated (Sephadex, Bio-Gel) or compact polyacrylamide gels. Conditions standardized for the determination of pI values of selected pH marker proteins proved inadequate for the predominant isoenzyme of horseradish peroxidase which requires an excessively long focusing time to attain the steady state. Carbon dioxide interferes with the determination of pI values >8.2 to 8.3. Thin-layer isoelectric focusing in a CO₂-free atmosphere followed by pH measurements also in a CO₂-free atmosphere, yields for alkaline marker proteins and the predominant peroxidase isoenzyme, pI values in excellent agreement with these found by the density gradient technique. The isoionic point of the predominant peroxidase isoenzyme determined by ion exchange desalting is identical with the isoelectric point found by density gradient and thin-layer isoelectric focusing in a CO₂-free atmosphere.     

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.     

Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.

Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

Radioimmunofixation of human ferritin following serum isoelectric focusing

Radioimmunofixation of human ferritin following isoelectric focusing of serum was developed to study the microheterogeneity of this protein in native serum without previous purification or concentration. This method requires only 2-10 microliter of serum and can be used with levels of ferritin as low as 10 micrograms/l. In this way, the extensive microheterogeneity of this protein was revealed, since in some cases it produced as many as 35 bands with isoelectric points in a pH range of 4.95-5.9. Very different isoelectric focusing patterns (spectrotypes) of ferritin were observed during the investigation of pathological sera. The high sensitivity of this technique makes it useful for the investigation of serum ferritin in diseases involving modifications of the metabolism of this protein.     

Isoelectric focusing and isoelectric points of bovine liver histones

A technique for isoelectric focusing of total histones in very narrow pH gradients is described. The isoelectric focusing was performed in 5% acrylamide gels at the pH range 9–11 in long quartz tubes (24 cm) in a nitrogen atmosphere. The total bovine liver histones separated into five main fractions which were identified as H1, H3, H2B, H2A, and H4 histones, and their apparent isoelectric points were determined. The main fractions were further divided into several subfractions, the maximal number of bands being 12. The isoelectric point for H1 histone in 6.25 m urea solution in the presence of a nitrogen atmosphere was 8.90, and the corresponding values for H3, H2B, H2A, and H4 histones were 9.80, 9.90, 10.10, and 10.25, respectively. The focusing technique described here has a high resolution, reproducibility, and sensitivity. The technique can be used for preparative and quantitative analysis and for studies on specificity and developmental changes of histones.     

Isoelectric focusing studies on the neutral 5′-nucleotidase from wistar rat hippocampus: Evidence for the absence of isoenzymes

Summary The pattern and some substrates characteristic of the rat brain 5-nucleotidase were studied using the isoelectric focusing technique, which revealed that the enzyme is present in a single form in hippocampus extracts. An alkaline phosphatase, which is also able to split nucleoside monophosphates, is not active at neutral pH values. The isoelectric points were found to be 6.4±0.1 for the specific 5-nucleotidase and 6.8±0.1 for the phosphatase.The present research paper was supported by grants from the Ministerium für Hoch- und Fachschulwesen der DDR     

Characterisation of ionogenic groups and estimation of the net negative electric charge on the surface of cells using natural pH gradients

The technique of isoelectric focusing has been extended to the study of the cell surface. A few tumour cell types and normal liver cells have been examined and are found to have characteristic isoelectric points. The isoelectric point of a cell, it is shown, provides information about the ionogenic groups present on its surface. The net electric charge borne by cells at their isoelectric points can be used to predict their electrophoretic mobilities in buffers at physiological pH and ionic strengths.     

Molecular heterogeneity of secretory IgA anti-DNP antibodies

Evidence is presented that demonstrates that the microscale sucrose isoelectric focusing technique can be used to assess the molecular heterogeneity of secretory IgA anti-DNP antibodies. The data indicate that substitution of the DNP group on the bacterial carrier, pneumococcus, can limit the structural complexity of secretory IgA anti-DNP antibodies. The data support the concept that factors governing secretory IgA antibody heterogeneity are analogous to those influencing the heterogeneity of serum IgG antibodies.     

Studies on the Xh antigen in human serum

Summary An antiserum against the Xh antigen in human serum was produced by injecting rabbits with pooled serum from healthy women and absorption of the immune serum with some male serums. The sedimentation coefficient of 12.2 S for the protein which carries this antigen was determined by density gradient centrifugation. The isoelectric point of 4.75–4.78 was measured by isoelectric focusing. The antigen can be demonstrated in 97% of the female and in 88% of the male serums. These characteristics do not fit any of the well studied serum proteins.This work was supported in part by NIH Program Project Grant HE-06285 from the National Heart Institute.