BSE: can we predict the future?

Prion diseases are transmissible neurodegenerative disorders of humans and animals. The prion protein (PrPc) gene is expressed to some extent in many cell types but principally in neurons. Normal PrPc may contribute in the protection of neurons and are protease sensitive. Abnormal prions consist of a post-translationally modified form of PrP, PrPsc, which is partly protease resistant. PrPsc is a protein with high resistance to inactivation by irradiation, heat and harsh chemical treatments. It is currently proposed that PrPsc is an infectious protein that propagates by inducing the normal PrPc to become the abnormal PrPsc. PrPsc cause transmissible spongiform encephalopathies (TSE), an unusual group of degenerative brain diseases that can be transmitted by inoculation or ingestion of diseased brain or other tissues. The human diseases occur in an inherited, acquired and sporadic forms. Transmission of prion diseases between species is limited by a species barrier, determined in part by the degree of sequence homology between the host PrP and inoculated PrPsc. The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom is a new disease that has affected over 160,000 cattle and has presumably arisen from dietary exposure to PrPsc from sheep with scrapie. Until shown otherwise we must assume that oral consumption of infectious BSE protein is a new factor for Creutzfeldt-Jakob (CJD) disease in man. This disease is a new variant of CJD (nvCJD) and has a different clinical picture. Early diagnostic markers to facilitate the diagnosis and screen blood and organ donors are not available. The control of the disease relies on the measures to eradicate the spread of BSE by banning the use of ruminant tissues in ruminant feed and slaughter and rendering procedures to ensure inactivation of prions of all infected animals. The control of nvCJD is based on reduction of exposure to BSE by banning a variety of tissues for consumption. A surveillance worldwide is increased for both BSE and nv-CJD and the WHO update regularly recommendations to limit the risk of transmitting the disease. The extent and size of the nvCJD will depend on different factors among others the dose of infectious material, the route of infection, the genetic susceptibility of the person. Therefore is not possible to predict how important the disease will be.     

Reversibility of scrapie inactivation is enhanced by copper

The only known difference between the cellular (PrPC) and scrapie-specific (PrPSc) isoforms of the prion protein is conformational. Because disruption of PrPSc structure decreases scrapie infectivity, restoration of the disease-specific conformation should restore infectivity. In this study, disruption of PrPSc (as monitored by the loss of proteinase K resistance) by guanidine hydrochloride (GdnHCl) resulted in decreased infectivity. Upon dilution of the GdnHCl, protease resistance of PrP was restored and infectivity was regained. The addition of copper facilitated restoration of both infectivity and protease resistance of PrP in a subset of samples that did not renature by the simple dilution of the GdnHCl. These data demonstrate that loss of scrapie infectivity can be a reversible process and that copper can enhance this restoration of proteinase K resistance and infectivity.     

Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells

The central causative event in infectious, familial, and sporadic forms of prion disease is thought to be a conformational change that converts the cellular isoform of the prion protein (PrPC) into the scrapie isoform (PrPSc) that is the primary constituent of infectious prion particles. To provide a model system for analyzing the mechanistic details of this critical transformation, we have previously prepared cultured Chinese hamster ovary cells that stably express mouse PrP molecules carrying mutations homologous to those seen in familial prion diseases of humans. In the present work, we have analyzed the kinetics with which a PrP molecule containing an insertional mutation associated with Creutzfeldt-Jakob disease acquires several biochemical properties characteristic of PrPSc. Within 10 min of pulse labeling, the mutant protein undergoes a molecular alteration that is detectable by a change in Triton X-114 phase partitioning and phenyl-Sepharose binding. After 30 min of labeling, a detergent-insoluble and protease-sensitive form of the protein appears. After a chase period of several hours, the protein becomes protease-resistant. Incubation of cells at 18 degrees C or treatment with brefeldin A inhibits acquisition of detergent insolubility and protease resistance but does not affect Triton X-114 partitioning and phenyl-Sepharose binding. Our results support a model in which conversion of mutant PrPs to a PrPSc-like state proceeds in a stepwise fashion via a series of identifiable biochemical intermediates, with the earliest step occurring during or very soon after synthesis of the polypeptide in the endoplasmic reticulum.     

Molecular analysis of bovine spongiform encephalopathy and scrapie strain variation

Five mouse scrapie strains, a mouse-passaged scrapie isolate derived from a field case in sheep in Germany, and 2 mouse-passaged bovine spongiform encephalopathy (BSE) isolates were analyzed by immunoblot in regards to banding patterns of proteinase K-digested pathologic prion proteins (PrPres). To obtain reliable results, the photo-imager technique was used for measurement of staining band intensities. Distinct and reproducible profiles were observed for the different strains or isolates. A British and a German BSE isolate were similar, suggesting the same source of infection. The German scrapie isolate resembled scrapie strain ME7, which has frequently been isolated from sheep scrapie in the past. In selected strains or isolates, no influence of the mouse lines used was observed on PrPres profiles, nor were brain region-specific differences apparent. This investigation suggests that PrPres glycotyping can be an invaluable tool for the in vitro differentiation of BSE and scrapie isolates.     

Prions and the prion disorders

One of us remembers sitting in a high school biology class in 1977 being taught about scrapie, a naturally occurring disorder of sheep. The teacher had no particular interest in agriculture, but was pointing out some peculiar characteristics of this disease as a biological curiosity on a wet Friday afternoon. The prion disorders captured the imagination of a range of biologists (including that teacher) well before the epidemic of bovine spongiform encephalopathy (BSE) and the appearance of a new variant of the human prion disease, Creutzfeldt Jakob disease (CJD), in the UK, because of their extraordinary biology and the unique properties of the infectious agent. We review the results of studies leading to a convergence of evidence that the causative infectious agent, the 'prion', is devoid of nucleic acid and is composed of an abnormal isoform of a host-encoded protein, the prion protein (PrP).     

Oral administration of (-)catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil

Prions diseases are fatal neurodegenerative disorders resulting from conformational changes in the prion protein from the normal cellular form, PrPC, to the infectious scrapie isoform, PrPSc. High resolution structures for PrPC are now available, and biochemical investigations are shedding light on the nature and determinants of the conformational transition. Together, these studies are beginning to provide a framework to describe structure-function relationships of the prion protein.     

Determination of the frequency and distribution of vascular and parenchymal amyloid with polyclonal and N-terminal-specific PrP antibodies in scrapie-affected sheep and mice

Brains from 17 histopathologically confirmed cases of scrapie, five of which had congophilic vascular amyloid, were stained immunohistochemically for prion protein (PrP) using a polyclonal antibody. Two clinically suspect but pathologically unconfirmed cases of natural sheep scrapie and the brains of four mice infected with the 111A murine scrapie strain were also examined. Selected sections containing amyloid were stained with each of two peptide antibodies which recognise the N-terminal amino acid residues which are lost following protease digestion of the disease-specific isoform of PrP. The mice infected with the 111A murine scrapie strain had large numbers of hypermature plaques. All the amyloid plaques from both natural sheep scrapie brains and experimental murine brains were heavily immunostained by the polyclonal and both peptide antibodies. In addition, disease-specific accumulations of PrP were detected in endothelial cells or in the intima of blood vessels of the cerebral cortex of sheep scrapie brains. The affected blood vessels were located in areas which otherwise lacked typical scrapie pathology. Vascular accumulations of PrP were also found in leptomeningeal and choroid plexus blood vessels. Vascular amyloid was found mainly in the neocortex. Vascular amyloid and disease-specific parenchymal accumulations of PrP were found in two sheep which showed clinical signs of scrapie but lacked its typical vacuolar pathology. These results show that the mature amyloid of scrapie is composed of, or contains a substantial proportion of, whole length PrP protein. Thus truncation of PrP is not essential for the aggregation of PrP into amyloid. The vascular amyloid of natural sheep scrapie originates from the accumulation and release of PrP from endothelial cells presumably following systemic scrapie infection. The topography of vascular amyloid distribution in Great Britain differs from that reported in the Netherlands. As amyloid deposition in mice is largely controlled by the strain of the infecting agent it is possible that the strain of the agent may influence vascular amyloid deposition.     

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.     

Prions

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.     

Transmissible spongiform encephalopathies in animals

Transmissible spongiform encephalopathies in animals are known for centuries. In particular scrapie in sheep and goats occurs worldwide; it spreads as a natural disease and is genetically controlled. Chronic wasting disease (CWD) in the United States (Wyoming and Colorado) also spreads as natural disease among free ranging and captive elk and mule deer. In contrast, transmissible mink encephalopathy (TME) of mink in fur producing farms is caused by contaminated feed; the source of this food contamination is still controversial. The only occurrence of a TSE in an avian species was reported from a flock of ostriches in a German zoo. The origin of the outbreak of bovine spongiform encephalopathy (BSE) in Great Britain could be traced back to feeding concentrates containing animal proteins contaminated with the agent of sheep scrapie. BSE was introduced into other countries, e.g. Switzerland and France, through the import of contaminated feeds from Great Britain. In addition, sporadic cases of TSE occurred in 10 other species, in particular domestic cats and zoo animals, e.g. antelopes and large cats. The diagnosis is based on the neuropathological examination of the brain as well as the demonstration of the disease specific protease resistant prion protein (PrPres). The first measures against the disease aim at eliminating the risk factors. The most important is the prohibition of feeding animal protein concentrates to ruminants. Thanks to this measure the incidence of BSE diminished remarkably. To protect consumers of beef products, in countries with BSE the potentially infectious organs of all cattle are confiscated at slaughter. Yet, in Great Britain this measure was introduced only at a point of time when BSE had already spread all over the country. Therefore, there is a strong probability of an exposition of consumers of beef with the BSE agent, which might have caused the new variant of Creutzfeldt-Jakob disease (nvCJD).     

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.     

Propagation of prion strains through specific conformers of the prion protein

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.     

Genetics of prion diseases and prion diversity in mice

Linkage of the prion protein (PrP) and scrapie incubation time genes in mice provided strong evidence for the central role of PrP in determining susceptibility to prion disorders. Considerable evidence now argues that the prion protein and incubation time genes are identical. The mouse prion protein gene (Prn-p) may act both quantitatively and qualitatively in modulating prion incubation time. Differences at positions 108 and 189 between PrP-A and PrP-B allotypes can place constraints on interaction between the normal cellular and the scrapie-specific isoforms of PrP (PrPC and PrPSc), although the supply of PrPC available for post-translational conversion to PrPSc can also influence incubation time. Results using transgenic (Tg) mice in studies on scrapie 'strains' or isolates suggest that incubation time characteristics of scrapie isolates can be explained by these two properties of PrP. The final section of this report discusses the novel finding that uninoculated Tg mice overexpressing wild-type (wt) PrP transgenes spontaneously develop a late-onset degenerative neuromyopathy, broadening the spectrum of prion diseases and providing new information on PrP function in both normal and pathological states.     

A mathematical model of the dynamics of scrapie in a sheep flock

A mathematical model is developed for the dynamics of an outbreak of scrapie in a single sheep flock with the aim of assisting the interpretation of field data. The model incorporates age structure of the sheep population, both horizontal and vertical transmission, genetic predisposition to infection, variable initial load of the infectious agent, and increasing infection load during an incubation period of the same order as sheep life expectancy. This leads to system of partial differential equations with respect to time, age and infection load. Numerical analyses using this model demonstrate that a scrapie outbreak is likely to be of long duration (several decades), will lead to reduction of scrapie susceptible allele frequency (but not to zero), and has different dynamics in homozygous and heterozygous susceptible sheep, even if these genotypes are equally susceptible, due to the different contributions of vertical infection to transmission to genotypes.     

Is the prion structure solved?

We report here on the current knowledge on the nature of the scrapie agent or prion. Several lines of evidence suggest that the abnormal isoform of prion protein (PrP) is crucial for scrapie infectivity while evidence that PrP is also a part of the entire particle of the scrapie agent (prion) is much weaker. There is no doubt, however, that conformational changes (transitions from alpha-helical into beta-pleated structures) of PrP underlay scrapie pathogenesis. In view of the notorious puzzling nature of the scrapie agent, the electron microscopic search for the ultrastructural correlate of it is still warranted. Thus, we discuss the nature of tubulovesicular structures (TVS), the only diseases-specific particles known so far and the association between TVS and PrP fibrils which was recently discovered.     

Recombinant scrapie-like prion protein of 106 amino acids is soluble

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.     

Prionoses--neurodegenerative diseases caused by prions, offectious proteinaceous molecules

Prionoses are a group of human and animal neurodegenerative diseases caused by prions, infectious pathogens that differ from bacteria, fungi, parasites, viroids, and viruses. Despite intensive searches over the past three decades, no nucleic acid has been found within prions and considerable experimental data argue that prions are composed exclusively of proteins (glycoproteins). Normal prion protein (PrPC) is encoded by a gene present in all nuclear cells of humans and other mammals but is constitutively expressed mainly in neurons. PrPC is protease sensitive and nonpathogenic but it can be modified to the pathological and protease resistant form designated PrPSC which is essential for infectivity. Prion diseases are manifested as infectious, genetic, or sporadic disorders and are also named as transmissible spongiform encephalopathies (TSE). TSE culminate with a progressive and fatal degeneration of the CNS. The human prionoses include Creutzfeldt-Jakob disease (CJD), kuru, Gerstman-Str?ussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In mammals, more than 15 different species have been described to suffer from prion disorders till now. Scrapie of sheep and goats is the oldest and the most studied of the prion diseases. Bovine spongiform encephalopathy (BSE) and transmissible mink encephalopathy are thought to result from the feeding of scrapie-infected animal products, whereas BSE has been identified in transmission to mice, domestic cats, two exotic species of ruminant, and monkey. More than 20 cases of clinically and pathologically atypical form of CJD, referred to as "new variant" CJD (vCJD) have been recognized in unusually young people in the United Kingdom. There is a strong evidence that the same prion strain is involved in both BSE and vCJD. It suggest the breaking of species barrier which results by spreading of BSE to humans, putatively by dietary exposure. Understanding the function of prion proteins and their modification to the pathological form may give new insight into the etiologic and pathogenic mechanisms also other diseases caused by aberrant proteins, including Alzheimer' disease, amyotrophic lateral sclerosis, and Parkinson's disease. (Tab. 4, Fig. 3, Ref. 76.)     

Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells

There is currently no effective therapy for human prion diseases. However, several polyanionic glycans, including pentosan sulfate and dextran sulfate, prolong the incubation time of scrapie in rodents, and inhibit the production of the scrapie isoform of the prion protein (PrPSc), the major component of infectious prions, in cultured neuroblastoma cells. We report here that pentosan sulfate and related compounds rapidly and dramatically reduce the amount of PrPC, the non-infectious precursor of PrPSc, present on the cell surface. This effect results primarily from the ability of these agents to stimulate endocytosis of PrPC, thereby causing a redistribution of the protein from the plasma membrane to the cell interior. Pentosan sulfate also causes a change in the ultrastructural localization of PrPC, such that a portion of the protein molecules are shifted into late endosomes and/or lysosomes. In addition, we demonstrate, using PrP-containing bacterial fusion proteins, that cultured cells express saturable and specific surface binding sites for PrP, many of which are glycosaminoglycan molecules. Our results raise the possibility that sulfated glycans inhibit prion production by altering the cellular localization of PrPC precursor, and they indicate that endogenous proteoglycans are likely to play an important role in the cellular metabolism of both PrPC and PrPSc.     

Carboxymethylation of thiol groups in ovalbumin: implications for proteins that contain both thiol and disulfide groups

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1% (w/v) sodium dodecyl sulfate at pH 8.2 with 5,5'-dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.     

A transient precursor of the HIV-1 protease. Isolation, characterization, and kinetics of maturation

Recently, the mechanism of autoprocessing of the protease (PR) of the human immunodeficiency virus type 1 from the model polyprotein, MBP-DeltaTF-PR-DeltaPol, which contains the protease linked to short native flanking sequences (DeltaTF and DeltaPol) fused to the maltose binding protein (MBP) of Escherichia coli, was reported (Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R., and Jerina, D. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7970-7974). According to this mechanism, intramolecular cleavage of the N-terminal strands of the dimeric MBP-DeltaTF-PR-DeltaPol protein leads to the formation of the PR-DeltaPol intermediate, which is subsequently converted to the mature protease by cleavage of the C-terminal strands. We now report the purification and characterization of the PR-DeltaPol intermediate and the kinetics of its processing to the mature protease. Unlike the MBP-DeltaTF-PR-DeltaPol precursor, PR-DeltaPol has proteolytic activity similar to that of the mature enzyme at pH 5.0. The pH rate profile for kcat/Km is similar to that of the mature protease above pH 4.0. Although the PR-DeltaPol is more sensitive than the mature protease toward denaturing reagents, both the enzymatic activity and the intrinsic fluorescence of PR-DeltaPol are linearly dependent on the protein concentration, indicating that the protein is largely in its dimeric form above 10 nM. In contrast to the first-order kinetics observed for the proteolytic reaction at the N terminus of the protease, the proteolytic reaction at the C terminus of the protease is second order in protein concentration. These results are discussed in terms of a mechanism in which the C-terminally located DeltaPol peptide chains are cleaved intermolecularly to release the mature protease.