A Protease Inhibitor of the Serpin Family Is a Major Protein in Carp Perimeningeal Fluid: I. Protein Purification and Characterization

Abstract: Two isoforms of a protease inhibitor of the serpin family (p62) have been purified from bighead carp perimeningeal fluid. Both isoforms migrate with an apparent molecular mass of 62 kDa on reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gels. Both proteins inhibited the activities of bovine trypsin, bovine chymotrypsin, and porcine pancreatic elastase. They also formed complexes with these proteases that were resistant to sodium dodecyl sulfate treatment. p62 exists in the extracts of all tissues examined, including brain, head kidney, kidney, liver, muscle, ovary, pituitary, and spleen. It is also present in serum, ovarian fluid, and milt as well as perimeningeal fluid. The protease inhibitor is a glycoprotein, and its carbohydrate moiety could be removed by endoglycosidase F. Because p62 resembles mammalian α₁-antitrypsin in many aspects, it is likely a fish equivalent of α₁-antitrypsin.     

Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Genetic control of PI and GC variants in the American Mink

Genetic polymorphism of the serum α-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 ).     

Structural features of archaebacterial and eukaryotic proteasomes

The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The molecule has a molecular mass of approximately 2000 kD and has a highly conserved structure in eukaryotes. The 26S proteasome is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S complex has C2 symmetry and is formed by four seven-membered rings of which the outer rings (-type subunits) are rotated by 25.7° relative to the inner rings while the inner rings (-type subunits) are in register. From a comparison of the activity and regulation of the 26S and 20S particles it can be deduced that the 20S particle contains the protease activity while the 19S complex contains isopeptidase, ATPase and protein unfolding activities. In this article we describe the structures of various proteasome complexes as determined by electron microscopy and discuss structural implications of their subunit sequences.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease

-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly¹⁵N- and ¹³C/¹⁵N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Subtilases: the superfamily of subtilisin-like serine proteases.

Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.     

Evidence for PDZ domains in bacteria, yeast, and plants.

Several dozen signaling proteins are now known to contain 80-100 residue repeats, called PDZ (or DHR or GLGF) domains, several of which interact with the C-terminal tetrapeptide motifs X-Ser/Thr-X-Val-COO- of ion channels and/or receptors. PDZ domains have previously been noted only in mammals, flies, and worms, suggesting that the primordial PDZ domain arose relatively late in eukaryotic evolution. Here, techniques of sequence analysis-including local alignment, profile, and motif database searches-indicate that PDZ domain homologues are present in yeast, plants, and bacteria. It is suggested that two PDZ domains occur in bacterial high-temperature requirement A (htrA) and one in tail-specific protease (tsp) homologues, and that a yeast htrA homologue contains four PDZ domains. Sequence comparisons suggest that the spread of PDZ domains in these diverse organisms may have occurred via horizontal gene transfer. The known affinity of Escherichia coli tsp for C-terminal polypeptides is proposed to be mediated by its PDZ-like domain, in a similar manner to the binding of C-terminal polypeptides by animal PDZ domains.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.