Pi Variants Research Articles

Alpha 1-Antitrypsin (AAT) deficiency and ANCA-positive systemic vasculitis: genetic and clinical implications.

A high incidence of alpha 1-antitrypsin (AAT) deficiency has been reported in patients with C-ANCA systemic vasculitis in association with antibodies against proteinase-3 (PR3). To clarify the role of AAT deficiency in the acute vasculitic process as well as in progression of the disease, we studied 84 patients with either C-ANCA or P-ANCA vasculitis with special reference to: (a) the AAT gene, (b) the phenotypic (Pi) variants and (c) the serum levels during both acute illness and remission. The PiZ gene was found in six patients (8% vs. 1.5% controls) irrespective of the type of autoantibodies (C-ANCA vs. P-ANCA). All PiZ patients displayed the ability to raise their AAT serum levels up to the normal range during acute illness. In contrast, 24 patients with the PiM phenotype presented low AAT serum levels during acute illness. In all these patients, the AAT levels returned to normal values during the remission. Low AAT levels were associated with low levels of C-reactive protein (PCR) (P < 0.001), with a less severe renal involvement or a minor risk of death, and, in one tested patient, with a novel point mutation (TCGA-->TCAA) at the enhancer-promoter region of the AAT gene. Low AAT serum levels did not correlate with either type/titre of autoantibody or distribution/severity of the vasculitis process. In the case-control study, high AAT levels emerged as a major determinant of progression towards end-stage renal failure [odds ratio 3 (95% CI 1.1-8.4)]. These results indicate: (a) a high incidence of the PiZ gene of AAT in systemic vasculitis irrespective of the type of autoantibodies; (b) a novel form of AAT deficiency associated with the normal PiM phenotype becoming manifest only during acute illness; (c) dysregulation of the acute-phase response affecting selectively AAT or both AAT and PCR; (d) correlation between low plasma levels of AAT and less severe renal involvement or risk of death.

Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis

Fis protein is shown here to bind to 10 sites in the gamma origin of plasmid R6K. The Fis-binding sites overlap all the previously identified binding sites in the gamma origin for the plasmid-encoded pi initiator protein and three host-encoded proteins, DnaA, integration host factor, and RNA polymerase. However, the requirement of Fis for R6K replication depends on the use of copy-up pi-protein variants and, oddly, the antibiotic resistance marker on the plasmid. In Fis-deficient cells, copy-up pi variants cannot drive replication of R6K gamma-origin plasmids carrying the bla gene encoding resistance to penicillin (Penr) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cmr). In contrast, R6K replication driven by wild-type pi is unaffected by the antibiotic resistance marker in the absence of Fis protein. Individually, none of these elements (copy-up pi, Fis deficiency, or drug markers) prevents R6K replication. The replication defect is not caused by penicillin in the medium or runaway replication and is unaffected by the orientation of the bla gene relative to the origin. Replication remains inhibited when part of the bla coding segment is deleted but the bla promoter is left intact. However, replication is restored by insertion of transcriptional terminators on either side of the gamma origin, suggesting that excess transcription from the bla gene may inactivate replication driven by pi copy-up mutants in the absence of Fis. This study suggests that vector sequences such as drug markers may not be inconsequential in replication studies, as is generally assumed.

Existence of phosphorylated and dephosphorylated forms of cytosolic thymidine kinase (TK1)

In this study we examine whether different TK1 variants of pI 6.9 and 8.3 found by isoelectric focusing gel electrophoresis (IFE) reflect just a phenotype difference due to phosphorylation modifications or have a real phenotypic background. The phosphorylation degree of purified TK1 variants was analyzed by determining the changes in the pI values after treatment with alkaline phosphatase, using IFE. The genetic origin of the two TK1 variants was studied by determining their mol wt. by means of SDS-gelelectrophoresis. Furthermore, the subcellular distribution of the two TK1 variants was also studied. Alkaline phosphatase treatment changed the pI value of purified TK1 from 6,9 to 8.3. No change in the pI value was found when purified TK1 corresponding to pI 8.3 was treated in the same way. Similar results were obtained when treated a cytosolic fraction with alkaline phosphatase. Antibody raised against the C-terminal part of human TK1 only recognized the dephosphorylated TK1 variant corresponding to pI 8.3. There was no difference in the molecular weight between the two TK1 variants. Thus, we concluded that the TK1 variants corresponding to pI 6.9 and 8.3 are of the same genetic origin, but consist of phosphorylated and dephosphorylated forms.

Size and charge heterogeneity of pituitary and plasma prolactin in the male rat.

Although pituitary prolactin (PRL) exhibits size and charge variability, questions concerning the structural heterogeneity and biological potency of hormonal forms secreted in vivo remain. In the present studies, monomeric PRL in male rat pituitaries and plasma was fractionated by Sephacryl S-100 size exclusion chromatography and aqueous chromatofocusing to resolve size and charge forms under conditions compatible with optimum preservation of biological activity. Individual hormonal variants were subsequently evaluated for their ability to stimulate the growth of PRL receptor-bearing rat lymphoma cells in vitro. Pituitary elution profiles contained several cross-reactive size variants ranging from 30.4 to 21.5 kD in M(r); major hormonal peaks were eluted at 25.6, 24.3, and 23.6 kD. Multiple size forms of PRL were also detected in plasma profiles, with predominant peaks eluting between 26.5 and 21.5 kD M(r) in size. Mean B/I ratios, established as an index of relative biopotency, varied significantly between size variants obtained from pituitary and plasma. Pituitary PRL size variants of 27.1, 24.3, and 21.5 kD exhibited greatest potency in the in vitro bioassay, whereas the 25.6- and 23.6-kD forms were least potent under these conditions. Of the PRL size variants detected in peripheral plasma, those of 24.3 and 21.5 kD size were characterized by highest mean B/I ratios. Pituitary 24.3-kD PRL was chromatofocused as five charge variants of pI 5.34, 5.31, 5.26, 5.20, and 5.14; only some of these isomers are apparently secreted in vivo, since pI values for plasma charge isomers ranged from 5.26 to 5.14. Charge isomers of pituitary 24.3-kD PRL exerted variable mitogenicity in the Nb2 in vitro bioassay. The highest mean B/I ratio was associated with the relatively basic 24.3-kD isomer of pI 5.31; the relative biopotency of more acidic charge isomers was progressively diminished with increasing acidic charge. In summary, the present findings show that both size and charge variants of male rat pituitary PRL exhibit differential biopotency in vitro, indicative of their functional heterogeneity. The demonstration of multiple hormonal forms in peripheral plasma suggests that the net biopotency of circulating PRL reflects the sum of activity of structurally and functionally diverse molecules.

Genetic control of PI and GC variants in the American mink.

Genetic polymorphism of the serum alpha-protease inhibitor (PI) and group-specific component (GC) in minks was revealed using one-dimensional polyacrylamide gel electrophoresis and immunoblotting. Two codominant alleles were identified at each of the two loci. The data ruled out the possibility of any linkage between the PI, GC and the coat colour gene Crystal (Cr).

Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K

The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein. Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype. We demonstrate here that these mutations prevent replication of a gamma origin core plasmid. To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants. We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.

Molecular heterogeneity of human chorionic gonadotropin in serum and urine from patients with trophoblastic tumors

Many studies on the structure and function of human chorionic gonadotropin (hCG) have relied on purified hCG preparations obtained from pregnancy urine. In the present studies, in order to demonstrate possible differences between the hCG species present in serum and those released into urine, we examined serum and urinary samples derived from patients with trophoblastic tumors and of pregnancy origin by sodium dodecyl sulfate electrophoresis, isoelectric focusing, and two-dimensional electrophoresis including immunostaining with a specific hCG antibody and densitometry of the protein bands. This type of analysis was presently feasible only in patients presenting with extremely high serum levels of hCG and was therefore limited to seven patients with testicular cancers, one woman with a hydatidiform mole, and one pregnancy sample. We found marked differences in the isoelectric focusing pattern between urinary and serum hCG samples, with the urinary hCG consisting of more alkaline pI variants than that present in the serum of the same patients. Tumor hCG differed from pregnancy hCG in that it contained more acidic variants, and this was true for urinary and serum-derived materials. In some tumor and pregnancy samples an hCG immunoreactive material of lower molecular weight than hCG itself was found. In conclusion, the present studies, extending previous findings on the microheterogeneity of hCG, indicate that serum and urinary-derived hCG may differ in the composition of the isoform spectrum, as does tumor and pregnancy hCG. Further, in some patients hCG immunoreactive molecules exist that differ markedly from hCG in size and charge. These observations suggest that, whenever possible, serum-derived hCG materials should be used to define the molecular structure of hCG and assess its biological activities.

Developmental changes in the isoelectric variants of rat hepatic hydroxysteroid sulphotransferase.

Major isoenzymes of androsterone-sulphating sulphotransferase (AD-ST) were isolated from liver cytosols of weanling and young adult female rats and their isoelectric properties were compared. On chromatofocusing the enzyme activity of young adults was eluted over a wider range of pH than was that of weanling rats. The activity at pH 7.8-7.2 (fraction I) is obvious at both ages, whereas the activity eluted over the pH 6.6-5.5 range (fraction II) is much lower in weanlings than in young adults. The AD-ST activities eluted in fractions I and II were separately purified by 3'-phosphoadenosine 5'-phosphate-agarose affinity chromatography at both ages. Two-dimensional gel electrophoresis of the isolated enzyme revealed several subunits with distinct pI values, but with the same molecular mass, namely 30 kDa. The relative levels of the pI 6.7 and pI 7.2 subunits are high and the relative level of the pI 6.1 is low in fraction I. In fraction II, the levels of pI 6.1 and pI 6.7 subunits are high and the level of the pI 7.2 subunit is low. There is no significant difference in the relative levels of the pI variants in each fraction between weanlings and young adults. The N-terminal amino acid sequences of the pI variants are identical within the area determined, irrespective of animal age or pI values. These results demonstrate that the pI variants of AD-ST are derived from the same precursor by post-translational modification or that they are products of closely related, but distinct, genes. The pI 6.1 and 6.7 subunits presumably increased during the development from the weanling stage to adulthood, resulting in the increase in acidic form(s) of AD-ST (fraction II) in adult females.

Variants of human chorionic gonadotropin from pregnant women and tumor patients recognized by monoclonal antibodies.

In biological fluids, hCG and its free alpha- (hCG alpha) and beta-subunits (hCG beta), occur in multiple forms. These various forms differ at the molecular level primarily in glycosylation, but also differ in protein backbone modifications corresponding to the urinary low molecular weight fragment of the hCG beta-subunit (beta-core fragment). This microheterogeneous nature can be demonstrated by isoelectric focusing in which variants are separated into bands with different isoelectric points (pI). To determine whether such isoelectric variants differ in antigenicity and consequently might escape immunoassay detection due to overspecificity of monoclonal antibodies (MCA), urinary pregnancy hCG (NIH, CR123) and tumor hCG preparations, such as a tumor-specific acidic variant of hCG (hCGav) and the hCG beta-core fragment, were separated by isoelectric focusing in the absence or presence of 8 M urea, or by sodium docedyl sulfate-polyacrylamide gel electrophoresis and enzymatically immunostained using an MCA panel directed against 17 different hCG epitopes. MCA against 14 different epitopes accessible on holo-hCG recognized all pI variants of pregnancy holo-hCG or tumor-derived hCGav, as was true for the three MCA recognizing epitopes hidden on holo-hCG but accessible on the free subunits after hCG dissociation by urea. We conclude that each individual pI-isoform of holo-hCG and its free subunits expresses the entire set of epitopes recognized by our MCA panel. The carbohydrate moieties that form a biochemical basis for hCG heterogeneity seem to be neither of major antigenic relevance, nor are they structurally related to any particular epitope. Thus, various glycosylation forms of hCG, hCG alpha, hCG beta, and hCG beta-core in normal as well as in pathological samples should safely be detectable and measureable by immunoassays employing MCA with appropriate subunit specificity.

Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni.

Our laboratories previously isolated a putative extracellular or membrane-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene, designated a signal peptide-containing (SP) Cu/Zn-SOD, from Schistosoma mansoni. SOD activity was thus investigated throughout the life cycle of S. mansoni and found in all stages: eggs, miracidia, cercariae, schistosomula, lung-stage worms, and adult worms. The adult worms had the highest SOD activity (53 +/- 9 nitrite units), which was five times higher than that of eggs or miracidia and twice as high as that of 3-h-old mechanically transformed schistosomula. Cu/Zn-SOD constituted over 95% of the total SOD activity found in S. mansoni, compared with that of Mn-SOD. Most of Cu/Zn-SOD specific activity was associated with a detergent-extractable fraction of the parasite. Isoelectric focusing gel electrophoresis analysis revealed that there were four major pI variants of Cu/Zn-SOD present in the adult worms. Only two of these Cu/Zn-SOD pI variants were present in the 3-h-old mechanically transformed schistosomula. Fast protein liquid chromatography gel filtration fractionation of adult parasite extract was carried out to correlate the SP Cu/Zn-SOD with the SOD activity by using anti-SP Cu/Zn-SOD monoclonal antibodies, which separated the immunoreactive gene product and the SOD activity into different fractions. Quantitative tissue fractionation also revealed a discordant distribution of the gene product compared with that of Cu/Zn-SOD activity. These results indicated the existence of another Cu/Zn-SOD(s) in the parasite. Purification of the Cu/Zn-SOD activity from the adult worms showed that it represented the two lower-pI variants found in both adult worms and 3-h-old schistosomula. Peptide sequence analysis of the purified Cu/Zn-SOD confirmed that there is a second form of Cu/Zn-SOD in the parasite.

Reliable phenotyping of alpha‐1‐antitrypsin by hybrid isoelectric focusing in an ultranarrow immobilized pH gradient

Genetically determined phenotypes of the highly polymorphic human alpha 1-antitrypsin were examined by hybrid isoelectric focusing in a narrow immobilized pH gradient. The chosen pH range from 4.45 to 4.75 was useful for identification and classification of the common PI M subtypes and a number of PI variants in the microheterogeneous regions of m6, m7, and m8. A high degree of resolution and an improved sharpness of PI bands was achieved with this excellent technique. It allowed the distinction of a new PI M variant, which has been designated M8, or Mingolstadt, according to the PI nomenclature. The pI difference of this mutant to the slightly cathodically located subtype M3 is approximately 0.001 pH unit. In addition, some common as well as rare phenotypes are presented.

The COOH terminus of several liver carboxylesterases targets these enzymes to the lumen of the endoplasmic reticulum.

To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.

P52 induction by cytochalasin D in rat kidney fibroblasts: Homologies between p52 and plasminogen activator inhibitor type‐1

Normal rat kidney (NRK) fibroblasts respond to the cell shape-modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52-kDa substrate-associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43-kDa "core" protein (p43), four 50-kDa species (p50-0,1,2,3), and at least seven distinct pI variants of the mature 52-kDa protein. A threshold of 2-4 microM CD was found to be necessary to augment p52 deposition into both the secreted protein- and saponin-resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD-treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type-1 (rPAI-1) and to PAI-1-like proteins of other species by comparative immunoprecipitation, 2-D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI-1 cytoplasmic mRNA abundance, using the rPAI-1 cDNA probe pSS1-3, revealed an induction of rPAI-1 mRNA in NRK/CD cells which paralleled the increased protein production. CD-augmented p52(rPAI-1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape-associated metabolic processes and concomitant RNA synthesis.

Alzheimer disease proteins (A68) share epitopes with tau but show distinct biochemical properties

Alz 50, a monoclonal antibody raised against Alzheimer brain homogenate, reacts with neurofibrillary tangles, microtubule-associated proteins tau, and Alzheimer brain proteins of molecular weight 70-60 kDa (A68). To study the relationship between A68 and normal human tau we compared the biochemical properties of these proteins and tested the reactivity of A68 with eight antibodies (Alz 50, Tau 60, Tau-2, Tau 14, Tau-1, Ab 636.7, NP14, Tau 46) that bind to various regions of tau molecule. On Western blots, all tau-reactive antibodies, except Tau-1, recognized A68. Pretreatment with alkaline phosphatase was required for the Tau-1 binding to A68. A68 consisted of three polypeptides of 68, 64, and 60 kDa, while tau contained 4-6 polypeptides of 50-65 kDa. A68 was less heterogenous than tau in the number of pI variants on two-dimensional gels. All A68 variants were more acidic (pI 5.5-6.5) than human tau (pI 6.5-8.5). Phosphatase treatment had only a minor effect on the pI and mobility of A68. Limited proteolysis of A68 with trypsin or chymotrypsin generated large fragments of 56-66 kDa (chymotrypsin) and 40-45 kDa (trypsin). While none of the fragments was recognized by Alz 50, the chymotryptic fragments were reactive with all the other tau antibodies, and the tryptic fragments were positive with five of the antibodies (Tau 14, Tau-1, Ab 636.7, NP14, and Tau 46). The peptide maps of A68 differed from that of tau in the number and the size of the peptide fragments. The differences in biochemical properties of these proteins and the sharing multiple epitopes suggest that A68 is a modified form of tau. The modification in part may be due to phosphorylation, although other changes rendering different isoelectrical properties and susceptibility to proteases need to be considered. The removal of the Alz 50 epitope by a cleavage of a 2-3 kDa fragment which does not contain the most C-terminal epitope (Tau 46) indicates that the Alz 50 epitope is located at the N-terminal periphery of the A68 molecule.

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L.

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M(r). The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.

A new alpha 1-antitrypsin allele PI Poki: isoelectric focusing with immobilized pH gradients as a tool for identification for PI variants.

A new rare genetic variant PI Poki in the protease inhibitor system found in two Japanese subjects, was compared with some PI P variants using several electrophoresis systems. Although this variant was slightly anodal to P by separator isoelectric focusing, it could be readily distinguished by isoelectric focusing with immobilized pH gradients: the mobility of Poki became cathodal to P. This method proved to be a powerful tool for identification of PI variants.

PI(alpha 1-antitrypsin) polymorphism in the Japanese: confirmation of PI*M4 and description of new PI variants.

PI phenotyping by separator isoelectric focusing (SIEF) was performed on a total of 1000 unrelated Japanese individuals from two different areas of Western Japan. The PI M1M4 subtype was observed together with the six common PI M subtypes. PI*M4 was confirmed to be present but rare in the Japanese. Several new PI variants were identified by comparison runs of each variant with previously reported genetic variants. The significance of treatment of serum with dithiothreitol (DTT) followed by iodacetic acid (IAC) in determination of PI variants is also described.

Elevated levels of a 17-kilodalton nuclear protein in carcinomas

The presence of a 17-kilodalton nuclear protein with pI variants of 6.7–6.9 in solid human malignant tumors is reported. This protein was more prominent in the carcinomas sampled compared to the sarcomas studied. Small amounts were detectable in placenta, but not in adult human liver, suggesting that the protein may be the product of a gene involved in normal differentiation.

The microheterogeneities of thyroxine-binding globulin and α1 protease inhibitor ( α1-antitrypsin) are interrelated

Addition of 125I-thyroxine to serum allows autoradiography for thyroxine-binding globulin microheterogeneity to be carried out after isoelectric focusing has been performed to display (by protein stain) the heterogeneous bands of the α 1-antitrypsin (PI) system. Comparison of the protein stain for PI with the autoradiograph for thyroxine-binding globulin indicates that these two systems are interrelated with the major bands of the PI system corresponding to the bands on the autoradiograph. This correspondence holds for PI variants other than the common M type and in particular it holds for the deficient Z type in which the autoradiograph for thyroxine-binding globulin is strikingly different from normal. We conclude that the major cause of microheterogeneity of TBG is due to an association with the PI system under the conditions of isoelectric focusing as normally performed. Precipitation experiments with antisera to PI and TBG suggest that the complex between these biologically important globulins may occur under conditions other than isoelectric focusing, but further work will be needed to examine this possibility.

Isoelectric focusing in immobilized pH gradients for the determination of the genetic Pi (α1‐antitrypsin) variants

AbstractThe genetic variants of the human protease inhibitor (Pi) system have been analyzed by isoelectricfocusing in immobilized pH gradients. With the use of a narrow pH range of pH 4.50 to 4.70 the six common PiM subtypes can be readily and reliably classified. The less common inherited Pi variants can be demonstrated most clearly in a gradient of pH 4.4 to 4.8. For the classification of PiZ a more basic pH gradient of 4.55 to 5.05 has been employed. Isoelectric focusing with immobilized pH gradients is also a useful tool for the study of the genetic variation in the Pi system. A number of problems to be adressed in future studies are discussed such as the delineation of further PiM subtypes, the detection of additional inherited heterogeneity, and the application of isoelectric focusing with immoblilized pH gradients in routine typing laboratories.