Isolation and partial characterization of rabbit plasma α1-antitrypsin

Abstract

Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.

Differences in lung glutathione metabolism may account for rodent susceptibility in elastase-induced emphysema development

“…7A), both control and 7-day BALF samples showed two ␣ 1 -AT immunoreactive bands in the range of 52-60 kDa, corresponding to the native ␣ 1 -AT described in rodents (23). As a result of elastase instillation, the two-band pattern for the native ␣ 1 -AT became more evident and, two new bands were observed.…”

Conformational differences between mouse contrapsin and .ALPHA.-1-antitrypsin as studied by ultraviolet absorption and circular dichroism spectroscopy.

“…The homologous protein has been purified from the monkey (Berninger and Mathis 1976), horse (Pellegrini and Fellenberg 1980), dog (Abrams et al 1978), rabbit (Koj et al 1978), and rat (Takahara et al 1980; Ikehara et al 1981).…”

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors

“…This is concluded from the observations that (i) mouse contrapsin, like RPI II, is inhibitory towards trypsin but does not affect the activity of chymotrypsin or elastase, (ii) in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis mouse contrapsin migrates as a molecule with an Mr of 64000, a value almost identical with that found for RPI II (65000), and (iii) neither inhibitor immunologically cross-reacts with cxl-PI. A number of mammalian sera, such as those of man (Yoshida & Mega, 1979), rabbit (Koj et al, 1978), dog (Abrams et al, 1978), horse (Pellegrini & von Fellenberg, 1980), mouse (Takahara & Sinohara, 1982) and rat (Rosenberg et al, 1976), have been reported to contain two or more forms of a1-PI, which can be separated by polyacrylamidegel electrophoresis. Mouse and rat, however are unique among them, since their two isoforms do not cross-react immunologically, whereas those from the other species are immunologically identical.…”

“…a1-PI has been isolated and characterized not only from man (Pannell et al, 1974) but also from plasma of several mammals, such as rabbit (Koj et al, 1978), mouse (Takahara & Sinohara, 1982) and rat (Rosenberg et al, 1976;Ikehara et al, 1981;Roll & Glew, 1981;Thisner & Rosengren, 1982 serum (Dahlmann et al, 1981), we used, in the present study, a new preparative ion-exchange medium (Duppel, 1982), together with two conventional steps, to purify a1-PI to homogeneity. In addition, two similar, but functionally distinct, proteinase inhibitors, tentatively designated RPI I and RPI II, were purified.…”