The Purification and Properties of Human α₁‐Antitrypsin (α₁‐Antiprotease), Variant Z

Abstract

After three stages of preliminary purification, variant Z was chromatographed on a DEAEcellulose column. Upon elution with a linearly increasing concentration of NaCI, variant Z was recovered in two separate peaks, the first of which contained 81 % and the second 19 % of the total.The preparation corresponding to the first peak was homogeneous by various criteria. The trypsin and chymotrypsin inhibiting capacities and the specific antigenic activity of the preparation were nearly the same as those of an authentic sample of variant M. Variant Z contained 8 or 9 more glycine residues than variant M, but no appreciable difference was found between their carbohydrate contents. By analytical isoelectrofocusing the isoinhibitors of purified variant Z overlapped with those in the plasma of the donor and were cathodal to, but partially overlapped with purified variant M. After desialysation, the overlap between the different variants became complete, but variant Z contained a larger proportion of cathodal and smaller proportion of anodal components than variant M. Both variants formed five distinct isoinhibitor-protease complexes after incubation with trypsin and chymotrypsin and the corresponding complexes in the different variants completely coincided.Over 20 different phenotypes of human al-antitrypsin can be distinguished by different electrophoretic and isoelectrofocusing methods (discussed in [I]). Some of the alleles of al-antitrypsin leading to a decreased plasma level of this protein, namely Piz (Pi standing for proteinase inhibitor) PiM Malton [lo] and PiM Duarte [ I l l attract special attention because of their medical as well was their theoretical implications.An outstanding discovery of the past few years in this field was the recognition of massive intrahepatic accumulation of al-antitrypsin in carriers of the Piz allele [12-151.The eel-antitrypsin in the serum of ZZ homozygotes is characterized by a slower (cathodal) than normal electrophoretic mobility . Several authors attempted to bring these observations to a common denominator by suggesting that both would arise decreased plasma level and decreased (cathodal) electrophoretic mobility of this protein [20,21]. This suggestion gained further credence by the observation that sialyltransferase activities in some scl-antitrypsindeficient members of a family were appreciably decreased [25].While this suggestion has had attractive features, on the whole it turned out to be untenable. As pointed out lately [26,27] the liver inclusion bodies in xlantitrypsin-deficient individuals are localized in the rough endoplasmic reticulum but have never been seen in the Golgi system of the hepatocyte, hence any possible alteration in the carbohydrate composition of this glycoprotein would be secondary to a primary defect of the peptide chain biosynthesis.In this work we purified variant Z from the plasma of a ZZ phenotype donor and compared it in various ways with purified variant M to gain insight into the nature of the defect leading to a1-antitrypsin deficiency. MA...

Z-type alpha 1-antitrypsin is less competent than M1-type alpha 1-antitrypsin as an inhibitor of neutrophil elastase.

“…The Z-type protein purified from plasma is also comprised of 394 amino acids and has carbohydrate side chains similar to that of the Ml molecule (4,27,49). The primary structure of the polypeptide differs from the MI(Val213) at two residues (Val23 to Ala213; Glu342 to Lys342) and from MI (Ala213) at one residue (Glu342 to Lys342) (48, [50][51][52].…”

Synthesis of abnormal immunoglobulins by hybridomas from autoimmune "viable motheaten" mutant mice.

“…This substitution disrupts an intramolecular salt bridge (23) that may be important for proper folding . However, Piz alleles from different individuals haveadditional mutations (40), and considerable molecular heterogeneity in the c«,-antitrypsin proteins from different Piz/Piz individuals has been reported (13,25) . Thus it is not unlikely that the a,-antitrypsin genes from different individuals have additional or different mutations that cause similar disruptions in tertiary structure of the protein and deposition of a,-antitrypsin in inclusion bodies .…”

Isolation and partial characterization of rabbit plasma α1-antitrypsin

“…A major proteinase inhibitor of mammalian blood plasma present in the a-globulin fraction and known as ax-antitrypsin has been isolated in a pure state from human blood by a number of investigators (Crawford, 1973;Kress & Laskowski, 1973;Pannell et al, 1974;Glaser et al, 1975;Miller et al, 1976;Saklatvala et al, 1976;Hercz & Barton, 1977). This glycoprotein occurs in multiple genetically determined variants, in some of which the deficiency in antitrypsin activity is responsible for pathological changes.…”