Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity

Herpes simplex virus (HSV) ribonucleotide reductase is formed by the association of two distinct dimeric subunits, R1 and R2. Attempts to purify either the HSV holoenzyme or its R1 subunit in their active form have been unsuccessful until now. The C terminus of the R2 protein being involved in the association with R1, the synthetic nonapeptide corresponding to this terminus, impedes the formation of the holoenzyme by competing with R2 for a critical site on R1. Based upon these observations, we developed an affinity chromatographic procedure to purify the R1 protein from HSV-1-infected baby hamster kidney cells. Specific binding of R1 to an affinity column made by linking the peptide HSV R2-(326-337) to Affi-Gel 10, followed by specific elution with an excess of an analogous peptide exhibiting a higher affinity for R1 yielded, in a single step, highly purified R1 protein. The purified R1 preparations contained approximately 95% of intact R1, the remaining 5% consisting of two R1 copurifying proteolytic breakdown products. The purified R1 protein exhibited a high reductase specific activity when mixed with an excess of the R2 subunit. Moreover, in vitro kinase assays revealed that the purified R1 protein of HSV-1 possesses an autophosphorylating activity also able to phosphorylate alpha-casein and histone II-S. The intrinsic protein kinase activity of HSV R1 is associated with its unique N-terminal domain which is absent from all other reductase subunits 1 and contains consensus motifs found in Ser/Thr protein kinases. A preliminary characterization of the kinase activity of the R1 protein of HSV-1 ribonucleotide reductase is presented.     

Affinity of Synthetic Peptides for the HSV-2 Ribonucleotide Reductase R1 Subunit Measured with an Iodinated Photoaffinity Peptide

The herpes simplex virus (HSV) ribonucleotide reductase comprises two nonidentical subunits, R1 and R2, which associate to form the active holoenzyme. A sensitive binding assay was developed to measure the affinity of inhibitory peptides for the HSV R1 subunit. The assay involved the use of a photoreactive radioligand [4′-azido-Phe³²⁸,3′,5′-¹²⁵I-Tyr³²⁹] HSV R2-(328-337), an analogue of the decapeptide Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu which corresponds to the C-terminal sequence (328-337) of the HSV R2 protein. As the radioligand binds covalently to the HSV R1 subunit upon uv irradiation, the affinity of peptide inhibitors can be easily determined by measuring their ability to compete with this highly specific binding. The method, which did not require any pure preparation of R1, was tested at 25 and 4°C and showed a significant increase in the affinity of the peptide inhibitors at 4°C. The relative affinity of these peptides was in agreement with their relative potency to inhibit reductase activity. The affinity of R2 subunit for R1 was also determined, and an IC₅₀ of 0.05 μM was measured. Altogether, this assay represents a precise and reliable tool with which to study more potent HSV ribonucleotide reductase peptide inhibitors, and the method could be applied to the study of other protein-protein and peptide-protein interactions.     

Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein

Proteolytic degradation of oxidatively damaged [3H] bovine serum albumin [( 3H]BSA) was studied during incubation with cell-free erythrocyte extracts and a wide variety (14) of purified proteases. [3H]BSA was pretreated by exposure (60Co radiation) to the hydroxyl radical (.OH), the superoxide anion radical (O2-), or the combination of .OH + O2- + oxygen. Treated (and untreated) samples were dialyzed and then incubated with erythrocyte extract or proteases for measurements of proteolytic susceptibility (release of acid-soluble counts). Both .OH and .OH + O2- + caused severalfold increases in proteolytic susceptibility (with extract and proteases), but O2- alone had no effect. Proteolytic susceptibility reached a maximum at 15 nmol of .OH/nmol of BSA and declined thereafter. In contrast, proteolytic susceptibility was still increasing at an .OH + O2-/BSA molar ratio of 100 (50% .OH + 50% O2-). Degradation in erythrocyte extracts was conducted by a novel ATP- and Ca2+-independent pathway, with maximal activity at pH 7.8. Inhibitor profiles indicate that this pathway may involve metalloproteases and serine proteases. Comparisons of proteolytic susceptibility with multiple modifications to BSA primary, secondary, and tertiary structure revealed a high correlation (r = 0.98) with denaturation/increased hydrophobicity by low concentrations of .OH. Covalent aggregation reactions (BSA cross-linking) may explain the declining proteolytic susceptibility observed at .OH/BSA molar ratios greater than 20. Protein denaturation may also have caused the increased proteolytic susceptibility induced by .OH + O2- + O2, but no simple correlation could be obtained. Results with .OH + O2- + O2 appear to have been complicated by direct BSA fragmentation reactions involving (.OH-induced) protein radicals and oxygen. These data indicate a direct and quantitative relationship between protein damage by oxygen radicals and increased proteolytic susceptibility. Oxidative denaturation may exemplify a simple, yet effective inherent mechanism for intracellular proteolysis.     

Susceptibility of varicella-zoster virus to thymidine analogues

Ten strains of varicella-zoster virus (VZV) were tested for susceptibility to 17 nucleoside analogues by a plaque reduction assay using human embryonic lung fibroblast cells. The compounds employed were 5-substituted arabinosyluracils and 2'-deoxyuridines, 2'-fluoro-arabinosylpyrimidines (F-araPyr) and acyclovir. In terms of the 50% plaque reduction dose (PD50), 4- to 40-fold difference were found between the 10 strains of VZV in susceptibilities to each compound. VZV was highly susceptible to 5-halogenovinyl-arabinosyluracils (XV-araUs); the PD50 values of these compounds were less than 0.001 micrograms/ml. VZV was much more susceptible than herpes simplex virus (HSV) type 1 to XV-araUs, but less susceptible than either HSV type 1 or type 2 to 5-ethyl-2'-deoxyuridine, 5-ethyl-arabinosyluracil and acyclovir.     

Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation

Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides. Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase. Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography. This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases.     

Efficacy of methylenecyclopropane analogs of nucleosides against herpesvirus replication in vitro

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC50 of 2.1 +/- 0.6 microM, compared with a mean EC50 of 3.9 +/- 0.8 microM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC50 values of 6.0 +/- 5.2 mciroM and <2.4 +/- 1.5 microM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC50 of 3.1 +/- 1.7 microM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Potent and selective MC-4 receptor agonists based on a novel disulfide scaffold

Extensive structure-activity relationship studies utilizing a beta-MSH-derived cyclic nonapeptide, Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (3), led to identification of a series of novel MC-4R selective disulfide-constrained hexapeptide analogs including Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (12). The structural modifications associated with profound influence on MC-4R potency and selectivity were ring size, ring conformation, and the aromatic substitution of the D-Phe7. These cyclic peptide analogs provide novel and enhanced reagents for use in the elucidation of melanocortin-4 receptor-related physiology, and may additionally find application in the treatment of obesity and related metabolic disorders.     

Differential Intracellular Compartmentalization of Herpetic Thymidine Kinases (TKs) in TK Gene-Transfected Tumor Cells: Molecular Characterization of the Nuclear Localization Signal of Herpes Simplex Virus Type 1 TK

The thymidine kinases (TKs) of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) were expressed in human osteosarcoma cells as fusion proteins with the green fluorescent protein (GFP), and their intracellular localizations were determined. The three TK-GFP fusion products were localized in different subcellular compartments of the transfected tumor cells. HSV-1 TK-GFP was localized exclusively in the nucleus, HSV-2 TK-GFP was predominantly found in the cytosol, while VZV TK-GFP was localized in both the nucleus and the cytosol. In support of these findings, we identified a nuclear localization signal (NLS) in the N-terminal arginine-rich region of HSV-1 TK that was absent in HSV-2 and VZV TK. The first 34 amino acids proved necessary for the specific nuclear localization of HSV-1 TK and, when added to the VZV TK-GFP gene construct, also sufficed to specifically target VZV TK-GFP to the nucleus. Further analysis of this NLS through site-directed mutagenesis revealed that the basic amino acid-rich nonapeptide ²⁵R-R-T-A-L-R-P-R-R³³ is of crucial importance in the nuclear targeting of HSV-1 TK. In particular, we revealed that the presence of the arginine residues at positions 25, 26, 30, 32, and 33 is obligatory for efficient NLS functioning, whereas arginine and histidine residues outside of the nonapeptide (i.e., residues R18, R20, and H22) did not change the functional properties of the NLS.     

Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis

Histatin 5 (Hst‐5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst‐5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single‐residue substitutions can significantly affect the proteolytic resistance of Hst‐5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single‐residue modifications can be combined in a single peptide, K11R–K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single‐residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap‐dependent effects on degradation. Both single‐ and double‐substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.     

The proteolytic enzymes as a highly sensitive criterion of effectiveness and safety of treatment

The effect of ozonated saline on proteolytic enzymes (trypsin, chymotrypsin, elastase, kallikrein, leucine aminopeptidase), inhibitors of proteolysis and lipid peroxidation (LPO) has been investigated. Injection of ozonated saline caused marked response of the proteolytic system. Low ozone doses did not cause activation of proteolytic enzymes, whereas high doses activated proteases, decreased the level of inhibitors of proteolysis (α₁-antitrypsin and α₂-macroglobulin) and stimulated accumulation of LPO products. Thus, analyses of proteolytic activity can be used as an indicator of effectiveness and safety of ozone therapy and other treatment programs.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells

Mixed-function oxidation of Escherichia coli glutamine synthetase has previously been suggested to mark the enzyme for intracellular degradation, and in vitro studies have demonstrated that oxidation renders the enzyme susceptible to proteolytic attack. In this study, the susceptibility of glutamine synthetase to degradation by purified proteases has been compared with the rate of degradation after microinjection into hepatoma cells. Upon exposure to an ascorbate mixed-function oxidation system the enzyme rapidly loses most of its activity, but further oxidation is required to cause susceptibility to extensive proteolytic attack either by a high-molecular-weight liver cysteine proteinase or by trypsin. The rate of degradation of biosynthetically 14C-labeled native and oxidized glutamine synthetase preparations after injection into hepatoma cells parallels their susceptibility to proteolysis in vitro. Native enzyme preparations and enzyme oxidatively inactivated, but not susceptible to extensive degradation by purified proteases, had similar intracellular half-lives; however, oxidized enzyme preparations that were susceptible to proteolytic breakdown in vitro were degraded almost ten times faster than the native enzyme within the growing hepatoma cells. These results suggest that the same features of the oxidized enzyme that render it susceptible to proteolysis in vitro are also recognized by the intracellular degradation system. In addition, they show that loss of enzyme activity does not necessarily imply decreased metabolic stability.     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Oxidative modification of glutamine synthetase. II. Characterization of the ascorbate model system

The first step in the proteolytic degradation of bacterial glutamine synthetase is a mixed function oxidation of one of the 16 histidine residues in the glutamine synthetase subunit (Levine, R.L. (1983) J. Biol. Chem. 258, 11823-11827). A model system, consisting of oxygen, a metal ion, and ascorbic acid, mimics the bacterial system in mediating the oxidative modification of glutamine synthetase. This model system was studied to gain an understanding of the mechanism of oxidation and of factors which control the susceptibility of the enzyme to oxidation. Availability of substrates and the extent of covalent modification of the enzyme (adenylylation) interact to modulate susceptibility of the enzyme to oxidation. This interaction provides the biochemical basis for physiologic regulation of intracellular proteolysis of glutamine synthetase. The oxidative modification requires hydrogen peroxide. While the reaction may involve Fenton chemistry, the participation of free radicals, superoxide anion, and singlet oxygen could not be demonstrated.     

Protease activities of rumen protozoa

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

The susceptibility of the glycoprotein from the purified (Na+, K+)-activated adenosine triphosphatase to tryptic and chymotryptic degradation with and without Na+ and K+.

Purified (Na+, K+)-activated adenosine triphosphatase ((Na+, K+)-ATPase, ATP phosphohydrolase, EC 3.6.1.3) has been subjected to trypsin and chymotrypsin hydrolysis. The glycoprotein is much more resistant to proteolysis than the large chain. This differential susceptibility to proteolysis is not due to differences in the number of trypsin or chymotrypsin sensitive bonds because the two subunits are equally susceptible to proteolysis after isolation by preparative gel electrophoresis in sodium dodecyl sulfate. It is also not due to steric "shielding" of the glycoprotein by the large chain or its proteolytic products: (1) The rate of digestion of the glycoprotein is not increased after 90% of the large chain is digested. (2) The majority of the large chain peptides are released into the supernatant upon degradation. It is concluded that the greater resistance of the glycoprotein to proteolysis is due to its native conformation. In the absence of the large chain, the susceptibility of the glycoprotein to tryptic degradation by K+ and Na+. The evidence suggests that this decreased susceptibility was due to conformational changes in the glycoprotein. These specific ligand effects on proteolysis of the glycoprotein suggests that the glycoprotein may participate in Na+ and K+ binding by (Na+, K+)-ATPase.     

Aminopeptidase resistant Arg-Gly-Asp analogs are stable in plasma and inhibit platelet aggregation.

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin alpha IIb beta 3) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH2 were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH2 with an acetylated or deleted alpha-amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I-fibrinogen binding to activated platelets (IC50 = 10-30 microM) and inhibited ADP induced platelet aggregation (IC50 = 10-30 microM). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.     

Site-directed mutagenesis of human ceruloplasmin:. production of a proteolytically stable protein and structure-activity relationships of type 1 sites

A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.