Bovine elastin

Ferrous ammonium sulfate from British Drug Houses, Liverpool, England; Bovine elastin and all other reagents from Sigma, London, England; 3,4(n)-³H proline and U-¹⁴C glycine were purchased from Perkin Elmer. Tritiated NaBH₄ was from Rc Tritec, Switzerland.
Patients from whom liver biopsies were obtained for diagnostic purposes served as controls. Approximately one-third of the tissue obtained was separated for enzyme determination, and the remainder was fixed in 10% formaldehyde for histological examination.
The specimens were quickly frozen within 90 sec in liquid nitrogen and then stored at −40 °C for a maximum period of 5 days before enzyme assays were performed.

Reduced ³H-bovine neck ligament elastin was prepared according to Takahashi et al. [47 (link)]. Ten milligrams of Bovine elastin (Sigma) suspension in distilled water was incubated with tritiated NaBH₄ (0.1 μCi/mg elastin). The reaction was terminated by the addition of acetic acid to pH 3.0. Elastin was removed by centrifugation, washed with distilled water, and lyophilized. A 0.5% (w/v) ³H-elastin suspension was prepared in 0.05 M Tris-HCl buffer pH 8.0. The incubation mixture contained 100 μL of this suspension, 100 μL of 0.05 M Tris-HCl buffer pH 8.0, and 100 μL of sample homogenate. After shaking for 60 min at 37 °C, the reaction was terminated by adding 50 μL diisopropylphosphorofluoridate (DFP) (final concentration 0.04 M). The mixture was centrifuged in a Beckman microfuge for 3 min, and 100 μL of the supernatant was counted for radioactivity. Bovine serum albumin blanks (0.05 mg/mL) were run simultaneously, and the enzyme activity was expressed as dpm per mg protein per hour.

Recombinant human tropoelastin was kindly provided by Anthony S. Weiss from the University of Sydney, Australia. Elastin-orcein, the chemical cross-linker bis(sulfosuccinimidyl) suberate (BS3 (link)), bovine serum albumin (BSA), and bovine neck ligament elastin isoform 1 (SwissProt accession number P04985–1, hereafter referred to as bovine elastin) were obtained from Sigma (USA). FAGLA was synthesized by ChinaPeptides Co., Ltd. (China). Recombinant pseudolysin was expressed in Escherichia coli and purified as previously described29 (link).