The complete amino acid sequence of human neutrophil elastase has been determined. The protein consists of 218 amino acid residues, contains two asparagine-linked carbohydrate side chains, and is joined together by four disulfide bonds. Comparison of the sequence to other serine proteinases indicates only moderate homology with porcine pancreatic elastase (43.0%) or neutrophil cathepsin G (37.2 %). In particular, many of the residues suggested to play important roles in the mechanism by which the pancreatic elastase functions are significantly changed in the neutrophil enzyme, indicating alternative types of binding with the human proteinase.Human neutrophils contain a battery of hydrolytic enzymes that normally are utilized for the degradation of foreign materials ingested as part of the phagocytic process (1). Although it is not completely clear whether such enzymes are also involved in tissue remodeling, this has been suggested as an alternate function (2). It is known that both neutrophil turnover and phagocytosis, themselves, do result in the leakage of enzymes into the extracellular milieu where they may cause extensive damage to connective tissue unless checked by controlling inhibitors (2). Indeed, a popular theory suggests that the development of pulmonary emphysema occurs as a result of insufficient levels of proteinase inhibitors (either locally or plasma-derived) whose primary functions are to inhibit these neutrophil enzymes (3). In particular, there is strong evidence that implicates neutrophil elastase as the proteinase most directly involved in abnormal lung connective tissue turnover. This is based on the fact that individuals devoid in the major controlling inhibitor of this enzyme, plasma a1-proteinase inhibitor, tend to develop obstructive lung disease (familial emphysema) much earlier than those with normal inhibitor levels (4).We have been interested in the potential function of both neutrophil elastase and cathepsin G in protein turnover. Both enzymes are capable of degrading a wide variety of substrates, including elastin, collagen, and proteoglycan, although it appears that elastase is more efficient in the turnover of such macromolecules (5, 6). Therefore, determination oftheir primary structures should be ofgreat value not only for understanding the mechanism by which each functions but also for developing specific inhibitors that might be useful in aiding in the control of their activities outside of the cell. This report describes the determination by conventional protein sequencing strategies of the primary structure of neutrophil elastase, a very basic glycoprotein that exists in a series of isoenzyme forms (7,8). In a separate report (9), a description of the primary structure of cathepsin G has been made by using a combination of recombinant DNA and protein-sequencing technology.MATERIALS AND METHODS Materials. Human neutrophil elastase was prepared from the granules of both normal and myeloid leukemia cells by affinity chromatography on Trasylol-Sepharose as described (1...

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

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