Alkaline Isoelectric Point Research Articles

Strategies for the enrichment and identification of basic proteins in proteome projects.

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.

Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.

A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.

Characteristic properties of proteins from pre-ecdysial cuticle of larvae and pupae of the mealworm Tenebriomolitor

Proteins extracted from the cuticle of pharate larvae and pupae of the mealworm Tenebrio molitor are more soluble at low temperatures than at higher temperatures, a behaviour characteristic of hydrophobic proteins. When the temperature of an unfractionated cuticular extract is raised from 4 to 25 °C the solution becomes turbid, droplets of a heavy, protein-rich phase are formed, which gradually settles, leaving an upper protein-poor phase, indicating that the aggregation process is a coacervation. The aggregation of the dissolved cuticular proteins is influenced by changes in temperature, pH, and ionic strength. The process has been studied by measuring development of turbidity in unfractionated cuticular extracts and in solutions of three purified proteins from Tenebrio pharate larvae and pupae (TmLPCP-A1a, TmLPCP-E1a, and TmLPCP-G1a), while temperature, pH or ionic strength of the solutions were varied. Protein aggregation was also studied by determination of changes in fluorescence intensity, when the hydrophobicity probe, 8-anilinonaphthalenesulfonic acid (ANS) was added to solutions of the cuticular proteins. Only when the protein solutions had developed a measurable turbidity was an increase in ANS-fluorescence observed, indicating formation of tightly packed clusters of hydrophobic amino acid residues during aggregation. The temperature range for aggregation depends upon protein concentration: the higher the concentration the lower and more narrow is the temperature range within which aggregation occurs. The tendency for the individual cuticular proteins to aggregate is most pronounced near their isoelectric points, and most of the cuticular proteins have alkaline isoelectric points. The influence of salts on the tendency of the proteins to aggregate varies among the proteins and depends upon how close they are to their isoelectric point. A solution containing both protein TmLPCP-A1a and TmLPCP-E1a becomes more turbid and develops a more intense ANS-fluorescence when warmed from 10 to 30 °C than corresponding to the sum of measurements performed on separate solutions of the two proteins, indicating that the two proteins interact during aggregation. The Tenebrio larval/pupal cuticular proteins are characterized by an abundance of hydrophobic amino acid residues, and especially their contents of alanine and proline are high. The behaviour of the cuticular proteins in solution resembles that of another hydrophobic protein, tropoelastin, and it seems reasonable to suggest that similar interactions govern the folding and aggregation of the peptide chains in the two types of proteins. The proline and alanine rich chain segments in the pharate cuticular proteins are suggested to form a series of beta-turns and to fold into a relatively open structure at low temperatures, giving water access to the hydrophobic residues and making the proteins water soluble. At increased temperatures the structure of the ordered water layer surrounding the hydrophobic groups breaks down, and the peptide chains tend to collapse into a more closed structure and to interact more tightly with hydrophobic regions in neighbouring molecules. In dilute solutions in the test tube this results in aggregation and precipitation of the proteins; in intact, pharate cuticle at ambient temperatures the proteins will preferably be in an aggregated, easily dissociated state. Accordingly, small changes in intercuticular pH and ionic strength can produce pronounced changes in the mechanical properties of unsclerotized solid cuticle by interference with protein interactions, in agreement with reports that some cuticles undergo plasticization during and/or immediately after ecdysis.

Regulation of serine-type exoproteinases by endogenous inhibitors present in exoantigens of the mycelial form of Paracoccidioides brasiliensis.

We have partially characterized some biochemical properties of exoproteinases secreted into culture medium by the mycelial form of Paracoccidioides brasiliensis, a dimorphic fungus that causes human disease in Latin America. Proteinase activity was analyzed in solid- and liquid-phase systems using zymography and Azocoll, respectively. Minimal or no gelatinase activity was observed by zymography in the crude filtrates among proteins with a relative mobility greater than 200 kDa. When the crude filtrate was fractionated by isoelectric focusing or ion exchange chromatography, we observed striking activation of gelatinases, both those of high apparent molecular mass and alkaline isoelectric points (pI), as well as those of lower molecular mass and acidic pI. The apparent high molecular mass gelatinases, pI 10, showed optimal activity at pH 7.0. They were totally inhibited by phenylmethylsulfonylfluoride and partially inhibited by incubation with previously neutralized fractions of pI 5.4 and 6.1. The latter inhibition could be reversed by exposure to 10% isopropanol. These results provide evidence of regulatory mechanisms controlling proteinase activity in secreted proteins. The principal mechanism appears to be the formation of reversible complexes with endogenous inhibitors.

Alkaline proteins of Bacillus subtilis: first steps towards a two-dimensional alkaline master gel.

The genomic sequence of Bacillus subtilis, which is the best studied Gram-positive bacterium, enabled us to obtain a theoretical two-dimensional (2-D) map, demonstrating that about one-third of this proteome has a theoretical alkaline isoelectric point (pI). This represents an important part of the entire proteome, which is not detectable in conventional 2-D gels (pH range 4-7). Sequence analysis revealed that 91% of the ribosomal proteins and a high amount of theoretical membrane proteins should be localized in the alkaline pH range requiring different protein extraction procedures. In order to find the pH range which gives the best resolution results for the alkaline proteins of B. subtilis, immobilized pH gradients (IPGs) with different pH ranges (pH 6-10, 6-11, 4-12, 9-12, and 3-10) were tested and optimized for IPG 4-12. Here we present a version of a first alkaline master 2-D gel for B. subtilis, which is a further complement of the already existing master gel (pH 4-7) in the Sub2D database. Almost 150 spots could be detected and 41 proteins have already been identified.

Alkaline proteins of Bacillus subtilis: First steps towards a two-dimensional alkaline master gel

The genomic sequence of Bacillus subtilis, which is the best studied Gram-positive bacterium, enabled us to obtain a theoretical two-dimensional (2-D) map, demonstrating that about one-third of this proteome has a theoretical alkaline isoelectric point (pI). This represents an important part of the entire proteome, which is not detectable in conventional 2-D gels (pH range 4—7). Sequence analysis revealed that 91% of the ribosomal proteins and a high amount of theoretical membrane proteins should be localized in the alkaline pH range requiring different protein extraction procedures. In order to find the pH range which gives the best resolution results for the alkaline proteins of B. subtilis, immobilized pH gradients (IPGs) with different pH ranges (pH 6—10, 6—11, 4—12, 9—12, and 3—10) were tested and optimized for IPG 4—12. Here we present a version of a first alkaline master 2-D gel for B. subtilis, which is a further complement of the already existing master gel (pH 4—7) in the Sub2D database. Almost 150 spots could be detected and 41 proteins have already been identified.

Interleukin 4 in Systemic Sclerosis: Not Just an Increase

Interleukin 4 in Systemic Sclerosis: Not Just an Increase

Characterization of the Proteins Comprising the Integral Matrix of Strongylocentrotus purpuratus Embryonic Spicules

In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

DNA polymerase betas from liver and testes of cherry salmon, Oncorhynchus masou: purification and characterization of DNA polymerase betas with acidic isoelectric points.

DNA polymerase betas from cherry salmon, Oncorhynchus masou, liver and testes were purified to near homogeneity, and no substantial differences between the enzymes were observed. The molecular weight of both enzymes, determined by SDS-polyacrylamide gel electrophoresis, was 39,000. The amino acid sequences of the N-terminus of the liver and testes enzymes were determined and compared with that of the rat enzyme. Of the N-terminal 30 amino acid residues of salmon liver DNA polymerase beta, 21 (70%) were identical to those of the rat enzyme sequence. However, unlike most eukaryotic DNA polymerase betas, the isoelectric points (pIs) of the DNA polymerase betas from salmon liver and testes were both estimated to be 6.2, which is significantly different from the alkaline isoelectric points (pI = 8.5-9.5) established for other highly purified vertebrate DNA polymerase betas. The cherry salmon DNA polymerase betas were still active at below 10 degrees C, compared with the rat enzyme.

Dodecyl maltoside detergent improves resolution of hepatic membrane proteins in two‐dimensional gels

This report describes the incorporation of an alkyl maltoside detergent in two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) sample lysis buffer in order to improve resolution of protein patterns separated by nonequilibrium pH gradient electrophoresis. Membrane-associated proteins with alkaline isoelectric points form horizontal streaks on two-dimensional electrophoretograms when solubilized with conventional nonionic detergent. Dodecyl maltoside enhances protein delipidation during solubilization and improves pattern resolution and protein mobility.

Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity

Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1–8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an M r at approx. 300 000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908–5912).

Purification and characterization of pectic enzymes from two races of Fusarium oxysporum f. sp. ciceri differing in virulence to chickpea ( Cicer arietinum L.)

Polygalacturonases (PG) and pectate lyases (PL) produced in vitro by races 0 and 5 of Fusarium oxysporum f. sp. ciceri have been purified by gel filtration followed by anion-exchange chromatography and, in some instances, chromatofocusing. Race 0, the least virulent race, produced three PG forms, designated PG I 0, PG II 0 and PG III 0, and one PL form, designated PL 0, whereas race 5, the more virulent race, produced only one PG form, designated PG 5, and two PL forms, designated PL I 5 and PL II 5. The molecular weights of the enzymes were estimated to be 44000 for PG I 0 and PG II 0 76000 for PG III 0 and PG 5, 25000 for PL 0, and 37000 for PL I 5 and PL II 5. By their mode of action, PG I 0 and PG II 0 were endo-enzymes, PG III 0 and PG 5 were exo-enzymes, and the three lyases were endo-enzymes showing, in contrast to the endo-PGs, very low or null activity on oligogalacturonides shorter than pentamers. With polygalacturonic acid as substrate, the values of pH optimum were 4·5 for all four PG forms, 9·5 for PL 0, and 10·5 for PL I 5 and PL II 5; the PL forms showed an absolute Ca 2+ requirement for activity. PG I 0, PL 0 and PL I 5 were cationic proteins with alkaline isoelectric points, whereas PG II 0, PG III 0, PG 5, and PL II 5 were anionic proteins with acidic isoelectric points. All the purified pectic enzymes were able to degrade chickpea cell walls although the degradative activity of the exo-PG 5 and the anionic endo-PL II 5 was notably lower than that of the remaining PG and PL forms. Degradability of wall preparations without ionically bound proteins was not related to host resistance or susceptibility. In contrast, walls containing ionically bound proteins from the chickpea susceptible cv. PV-24 were more extensively degraded by the various PG and PL forms than were similar preparations from the resistant cv. WR-315.

Purification and properties of a new ribosome-inactivation protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1–8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an M r of approx. 30 000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908–5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.

A peptidyl dipeptidase-4 from Pseudomonas maltophilia: Purification and properties

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.

Microheterogeneity of paraproteins. I. Diagnostic value of isoelectric focusing followed by immunoblotting

Isoelectric focusing (IEF) in thin-layer polyacrylamide gels followed by immunoblotting on nitrocellulose membranes is presently the most sensitive method in the routine detection of IgG paraproteins. With this technique, immunoglobulin class and light chain composition can be as reliably identified as in immunoelectrophoresis. The problem of firm adherence between IEF polyacrylamide gels and nitrocellulose membranes can be overcome by brief incubation in sodium dodecyl sulfate. After isoelectric focusing, IgG paraproteins display a characteristic pattern of limited electrophoretic heterogeneity. This pattern is easily recognized even in the few cases with a constant tendency to aggregate under IEF conditions and in the surprisingly high percentage of paraproteins with very alkaline isoelectric points in which it is altered due to a cathodal collection effect. It is independent of the total amount of IgG in serum and remains stable intraindividually over extended observation periods. On the other hand, there is a very high degree of interindividual variability while common paraprotein characteristics still remain recognizable.

Characterization of proteins from pharate adult wing cuticle of the migratory locust, Locusta migratoria

Thirteen of the more abundant proteins in pharate adult wing cuticle of the migratory locust, Locusta migratoria, have been purified to near homogeneity. Their amino acid compositions and electrophoretic titration curves were determined, showing that these proteins resemble the proteins from pharate adult locust body cuticle in being rich in alanine and/or glycine, containing few charged or polar groups, and having mainly alkaline isoelectric points. Glycine-rich proteins tend to be more dominating in wing cuticle than in sclerites, and one wing protein has a very high content of proline and valine. The specific pattern of proteins in locust wing cuticle is suggested to be important for giving the wings optimal mechanical properties.

A novel microtubule protein in the marginal band of human blood platelets.

A characterization is reported of the major cytoskeletal protein, called IEF (isoelectric focusing)-51K, of marginal band microtubule coils from human blood platelets (Kenney, D. M. and Linck, R. W. (1985) J. Cell Sci. 78, 1-22). IEF-51K is a unique biochemical species which is distinguishable from platelet and mammalian neuronal alpha-tubulin and beta-tubulin by 1) its faster mobility on discontinuous sodium dodecyl sulfate electrophoresis corresponding to an apparent Mr 51,000; 2) its more alkaline relative isoelectric point at pH 5.7 compared with that of alpha- and beta-tubulin at pH 5.3 and 5.5, respectively; 3) lack of coincidence in peptide maps prepared with chymotrypsin or Staphylococcus aureus V8 protease; and 4) lack of immunochemical cross-reactivity of polyclonal anti-IEF-51K with alpha- and beta-tubulin and of monoclonal anti-alpha-tubulin and anti-beta-tubulin with IEF-51K. In contrast to its chemical uniqueness, IEF-51K is tubulin-like in some of its properties. IEF-51K is localized in the marginal band of intact platelets by immunofluorescence; it undergoes cycles of microtubule disassembly/reassembly both in vitro and in vivo. Furthermore, IEF-51K was not extracted from isolated Taxol-stabilized marginal band microtubules by elevated NaCl concentrations (to 0.45 M), conditions that do not disrupt the polymeric structure of alpha- and beta-tubulin. These results indicate that IEF-51K together with alpha-tubulin and beta-tubulin are the major structural polypeptides of platelet marginal band microtubules. The unusual subunit composition of the platelet marginal band microtubule may be related to specialization(s) of microtubule structure and function in the marginal band coil of platelets.

Transition of beta-actinin isoforms during development of chicken skeletal muscle.

We examined by means of the immunoblotting technique the transition of beta-actinin isoforms during the development of the chicken from 5 day embryo to adult. As an antigen, beta-actinin was prepared from adult chicken breast muscle (pectoralis major) and polyclonal antibody was obtained by injecting undenatured beta-actinin into a rabbit. Immunoblotting examination of breast muscle at several stages of development (except 5 day embryo, in which the whole body minus the head and limbs was examined) showed that the species of beta-actinin subunits change during development: 1) beta I is already present in 5 day embryo, whereas beta II appears only after 9 days. 2) In 5 day embryo, we found, instead of beta II, a new subunit (designated beta III) that cross-reacts with the antibody, has the apparent molecular weight of 30,000 daltons and has a slightly alkaline isoelectric point compared with beta I. The content of beta III gradually decreased and beta III completely disappeared a week after hatching. Such a type of transition of the isoforms in beta-actinin subunits is similar to that observed in other muscle proteins. The transition of beta-actinin isoforms may correlate to the organization of an I-Z-I brush, especially to the length determination of thin filaments, because the developmental stage at which beta III disappears coincides with that at which the length of thin filaments is strictly determined.

The isoelectric focusing of creatine kinase variants: I. The heterogeneity of creatine kinase in human heart cytosol and mitochondria.

Creatine kinase isoenzymes in cytosolic and mitochondrial fractions from human cardiac tissues were studied by analytical and preparative isoelectric focusing (IEF), electrophoresis and immunoinhibition. Analytical IEF on agarose gels revealed many creatine kinase variants in human cardiac cytosol prepared by extraction with a hypotonic medium. The bands located at approximately pH 5.5 were shown to contain creatine kinase-MB and minute creatine kinase-BB bands by electrophoresis. Two bands which focused closely together in IEF (pH 6.85-7.0) showed an electrophoretic migration pattern similar to creatine kinase-MM. One of them (IP 6.85) showed a complete inhibition by anti-creatine kinase-M antibodies, whereas the other showed only 50% inhibition. Increasing the salt concentration of tris-HCl (0.1 mol/l) in the extraction medium resulted in additional creatine kinase variants. They were characterized by high alkaline isoelectric points and were not inhibited by anti-creatine kinase-M antibodies. These variants corresponded to two cathodic bands in electrophoresis. The treatment of washed mitochondria with phosphate buffer resulted in a release of mitochondrial variants with different isoelectric points, as shown by analytical IEF in agarose gels. The same pattern was obtained by using preparative IEF. Variants with high alkaline isoelectric points gave rise to two cathodic bands upon electrophoresis. These two bands resembled those present in cytosol after extraction with high salt concentration. No complete inhibition with anti-creatine kinase-M was observed in any of the eluates. The mitochondrial variants exhibited different affinities towards creatine phosphate and ADP. Variants with higher alkaline isoelectric points showed lower Km-values for these substrates than those with less alkaline isoelectric points.

The isoelectric focusing of creatine kinase variants: II. The heterogeneity of creatine kinase in human serum with normal and elevated catalytic concentrations.

An effective and reliable method for the quantitative estimation of creatine kinase-MB, creatine kinase-MM variants and mitochondrial forms of creatine kinase in serum is presented. The high resolving power of isoelectric focusing allows the use of tetrazolium salts and meldola blue for the quantitative measurement without interfering non-specific reduction. The addition of thiol compounds to the agarose medium increases the sensitivity of the method, due to the inhibition of sulfhydryl group oxidation, and prevents enzyme degradation, which is a possible cause of an artificial heterogeneity. Depending upon the type of muscle and the degree of cell damage, we found 3-4 creatine kinase-MM sub-bands in sera with activities below 80 U/l. At elevated creatine kinase activities 3-11 creatine kinase-MM sub-bands were found. The appearance of creatine kinase-MB in serum indicates that damage has occurred to certain organs, especially the cardiac muscle. An organ with moderate or massive cell damage could release, in addition to the sarcoplasmatic creatine kinase variants, other forms with more alkaline isoelectric points (mitochondrial creatine kinase). The presence of such bands in serum of patients correlates with poor prognosis. Besides the separation of creatine kinase-MM sub-bands, creatine kinase-MB, creatine kinase-BB and of macroforms 1 and 2, the advantage of this method is the detection of mitochondrial creatine kinase forms, which in cellulose acetate electrophoresis migrate with creatine kinase-MM.