Purification and kinetic characterization of human cytomegalovirus assemblin.
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Amino Acid Sequence↗
Biomedical subjects
G W Becker
Publications and source records attributed to G W Becker.
Characterization by electrospray mass spectrometry of human Ca(2+)-sensitive cytosolic phospholipase A2 produced in baculovirus-infected insect cells.
The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regulated phospholipase that is thought to initiate the production of inflammatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01% of total cellular protein) in various cells and tissues, we have used the baculovirus expression system to produce sufficient quantities of cPLA2 for structural and functional analysis. The cDNA for cPLA2 was cloned into a baculovirus expression vector and, upon infection of Spodoptera frugiperda Sf-21 cells with the recombinant virus, cPLA2 was produced at high levels (9% of total cellular soluble protein). Gel electrophoresis and immunoblot analysis demonstrated that the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombinant cPLA2, using electrospray mass spectrometry in conjunction with automated sequence analysis, confirmed the expected sequence and revealed two post-translational modifications of the protein, phosphorylation on at least one site, and acetylation of the N-terminal serine residue after removal of the initiating methionine. In spite of the presence of six potential N-glycosylation sites, there is no evidence that any of them is glycosylated. The baculovirus expression system should prove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.
Amino Acid Sequence↗
Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver.
The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.
Adenosine Triphosphatases↗
Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.
The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.
Amino Acid Sequence↗
Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro.
Amyloid beta peptide (A beta), the major protein constituent of senile plaques in patients with Alzheimer's disease, is believed to facilitate the progressive neurodegeneration that occurs in the latter stages of this disease. Early attempts to characterize the structure-activity relationship of A beta toxicity in vitro were compromised by the inability to reproducibly elicit A beta-dependent toxicity across different lots of chemically equivalent peptides. In this study we used CD spectroscopy to demonstrate that A beta secondary structure is an important determinant of A beta toxicity. Solubilized A beta was maximally toxic when the peptide adopted a beta-sheet conformation. Three of the four A beta lots tested had a random coil conformation and were weakly toxic or inactive, whereas the single A beta lot exhibiting toxic activity at low peptide concentrations had significant beta-sheet structure. Incubation of the weakly toxic A beta lots in aqueous stock solutions for several days before use induced a time-dependent conformational transition from random coil to beta-sheet and increased A beta toxicity in three different toxicity assays. Furthermore, the secondary structure of preincubated A beta was dependent upon peptide concentration and pH, so that beta-sheet structures were attenuated when peptide solutions were diluted or buffered at neutral and basic pH. Our data could explain some of the variable toxic activity that has been associated with A beta in the past and provide additional support for the hypothesis that A beta can have a causal role in the molecular neuropathology of Alzheimer's disease.
Amyloid beta-Peptides↗
Isolation and characterization of human liver cytochrome P450 2C19: correlation between 2C19 and S-mephenytoin 4'-hydroxylation.
In an effort to identify and characterize minor forms of human liver cytochrome P450, immunoblot analyses of microsome samples were developed with antibodies to various P450s that recognized multiple human P450s. Four P450s were recognized in immunoblot analyses of human liver microsome samples developed with an antibody previously demonstrated to specifically recognize rat 2B1/2. Three of these P450s were identified as 2A6, 2C9/10, and 2E1 and the fourth was termed P450UK. A monoclonal antibody to 2C9/10 recognized P450UK in addition to 2C9/10. In order to identify P450UK, it was purified and subjected to amino-terminal amino acid analysis. The amino-terminal sequence obtained for P450UK was identical to the sequence deduced from a cDNA encoding CYP2C19, thus identifying P450UK as 2C19. The relative levels of 2C19 were determined in 14 human liver microsome samples by quantitative immunoblot analyses developed with the anti-2C9/10 antibody. These analyses demonstrated that 2C19 was not detected in one sample and its levels varied 10.5-fold in the remaining samples. The levels of 2C19 were compared to the relative levels and catalytic activities of multiple human liver P450s. The levels of 2C19 and the ability of the samples to 4'-hydroxylate S-mephenytoin were found to strongly correlate (r2 = 0.79). In summary, this is the first demonstration of the expression of 2C19 at the enzyme level, and the correlation studies suggest that 2C19 plays a role in the 4'-hydroxylation of S-mephenytoin.
Amino Acid Sequence↗
Proteolysis of human growth hormone by rat thyroid gland in vitro: application of electrospray mass spectrometry and N-terminal sequencing to elucidate a metabolic pathway.
The present studies were designed to provide structural characterization of peptide metabolites of biosynthetic human growth hormone (hGH) formed by rat thyroid gland proteases in vitro. Electrospray ionization mass/spectrometry (ESI-MS) and N-terminal sequencing were used to characterize the peptide metabolites. The predominant enzyme in the thyroid gland preparations was a chymotrypsin-like serine protease which was biochemically similar to rat mast cell protease-I. Metabolic intermediates were formed by cleavage of hGH exclusively at Tyr/Phe/Leu-Xaa bonds. After a 5- or 45-min incubation of hGH with thyroid gland S9 pellet fraction, the majority of metabolites formed were two-chain variants of hGH having masses ranging from 16,002 to 22,143 Da. These metabolites were formed as a result of proteolysis in the large disulfide loop region of hGH in combination with processing at Tyr42-Ser43. Based upon the time-related appearance and structural characterization of these intermediates, a sequence of metabolic events is proposed. The initial event appears to be cleavage by the chymotrypsin-like protease between Tyr143-Ser144 to form a two-chain hGH. This intermediate is then cleaved between Tyr42-Ser43, liberating the N-terminal peptide, Phe1-Tyr42. Two other metabolites were generated as a result of the deletion of the peptides Lys140-Tyr143 and Ser144-Phe146 from the large loop region. The identification of similar metabolites truncated by a single amino acid at the carboxyl terminus indicated the action of a carboxypeptidase on these metabolic products. After a 4.5-hr incubation, the protease isolated from the S9 pellet fraction degraded hGH to > 20 small peptides, having masses < or = 2300 Da.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Comparative analyses of Serratia spp. outer membrane porin proteins.
Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunoblot and Southern DNA hybridization experiments and probed with antibody and DNA probes specific for the 41-kDa Serratia marcescens porin, to determine the extent of homology between Gram-negative porins. Immunoblot analyses performed using porin-specific rabbit sera and cell envelope preparations from these strains revealed that all strains produced at least one cross-reactive protein in the 41-kDa molecular weight range. Chromosomal DNA from each of the same strains was used in Southern analyses, probed with a 20-base-length oligonucleotide probe deduced from the N-terminal amino acid sequence of the 41-kDa Serratia marcescens porin. The probe hybridized to DNA from all of the Serratia species and six of the nine other enteric bacteria. Putative porin proteins from all the Serratia species were subjected to N-terminal amino acid sequencing and porin functional analysis using the black lipid bilayer method. All amino acid sequences were identical, with one exception in which an asparagine was substituted for an aspartic acid in Serratia rubidaea. All porins had very similar porin function (single channel conductance ranging between 1.72 and 2.00 nS). The results from this study revealed that a strong conservation exists among the Serratia porins and those produced by other enteric bacteria.
Amino Acid Sequence↗
Mechanisms involved in degradation of human insulin by cytosolic fractions of human, monkey, and rat liver.
The degradation of native and 125I-labeled human insulin (HI) was examined in the cytosolic fraction of human, monkey, and rat liver. The purpose of these studies was to provide a species comparison of the interaction of insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI) in the degradation of HI. Western-blot analysis with monoclonal antibodies indicated the presence of both IDE and PDI in the cytosolic fraction of human and monkey liver. In contrast, rat liver cytosol contained, detectable levels of IDE only. A species comparison of metabolic profiles was performed by fractionating peptide products with reversed-phase high-performance liquid chromatography. After a 60-min incubation, human liver cytosol degraded unlabeled HI into three major products. Two of these peptides coeluted with the products of the incubation of HI with purified rat liver PDI. The three peptides were isolated and determined by NH2-terminal sequence analysis to be intact A chain, B chain, and des(Phe1)-B chain. Human liver cytosol also formed 125I-A chain and 125I-B chain as major products when specifically labeled 125I-HI isomers were used as substrate. Significant proteolytic degradation was observed only when reactions with human liver cytosol were supplemented with Mn2+. In contrast, monkey and rat liver cytosol proteolytically degraded 125I-HI isomers to small peptide fragments. The rat and monkey metabolic profiles were similar to each other and to that observed with Mn(2+)-supplemented human liver cytosol. Proteolysis in monkey and rat was sensitive to inhibition by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Two monoclonal antibodies recognizing different epitopes on rat cytochrome IIB1 react with human IIE1.
To identify human cytochromes P450 (P450) in the CYP2B subfamily, 14 human liver microsomal samples were screened by immunoblots developed with monoclonal antibodies that recognized seven distinct epitopes on rat IIB1. Two of these antibodies recognized a protein in all of the samples. This protein was termed P450BE. Using video-imaging densitometry, the levels of P450BE were determined and compared with levels of other P450s. An excellent correlation was seen (r = 0.87) between P450BE and human IIE1. However, rat IIE1 did not react in immunoblot and enzyme-linked immunosorbant assays with the two anti-rat IIB1 monoclonal antibodies. As previously observed, the levels of IIE1 in the samples correlated well (r = 0.88) with the ability of these human liver microsomes to N-demethylate N-nitrosodimethylamine. The levels of P450BE also correlated well (r = 0.91) with the ability of the microsomes to N-demethylate N-nitrosodimethylamine. In addition, excellent correlations were obtained when the levels of P450BE and IIE1 were compared with the ability of the microsomes to O-deethylate ethoxycoumarin (r = 0.87 and r = 0.85, respectively). To identify the protein recognized by the anti-rat IIB1 antibodies, P450BE was purified from microsomes prepared from human liver D. Amino-terminal amino acid sequence analyses of P450BE revealed that the 18-amino acid sequence obtained matched the corresponding sequence of human IIE1. In addition, purified human IIE1 and P450BE migrated with the same apparent molecular weight in polyacrylamide gels. Furthermore, proteolytic maps of P450BE and IIE1, generated with two proteases, were found to be identical. Sequence alignments and antigenicity calculations identified three regions of rat IIB1 as likely candidates for the epitopes shared in common with human IIE1. In conclusion, this study indicates that caution must be taken when interpreting the results of immunochemical assays when species lines are crossed.
Adolescent↗
Catalytic hydrogenolysis of poly-iodinated recombinant human insulin-like growth factor II (IGF-II): a potentially useful method for the tritiation of IGF-II.
A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Mono- and di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the iodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.
Amino Acid Sequence↗
Proteolytic cleavage of human growth hormone (hGH) by rat tissues in vitro: influence on the kinetics of exogenously administered hGH.
The presence of several endogenous molecular forms of human GH (hGH), including proteolytically cleaved two-chain forms, has been proposed to be related to the diverse biological activity of hGH. The present study characterized hGH degradation in the rat to determine how peripheral metabolism may influence the kinetics and pharmacology of exogenously administered hGH. In vitro studies indicated that hGH was proteolytically degraded by thyroid gland and skeletal muscle, but not liver and kidney homogenates. The proteolytic activity, localized to the 9000 x g pellet fraction, was characterized as a chymotrypsin-like serine protease using class-specific inhibitors. N-Terminal sequencing of hGH peptides formed by the thyroid gland and skeletal muscle indicated that cleavage sites were almost exclusively at Tyr/Phe-Xaa bonds, with similar points of cleavage observed in the two tissues. Immunoreactive two-chain forms of hGH were also formed. The two-chain molecules had similar cleavage sites, but differed in apparent mol wt when analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To understand the potential significance of two-chain product formation, we compared the kinetics and degradation of hGH with those of a synthetic two-chain derivative of hGH (Des-1-8,135-145; 2-CAP). The in vitro tissue distribution of 2-CAP proteolysis was the same as that for hGH. The fragmentation pattern of 2-CAP was less complex when analyzed by reverse phase HPLC. The major peptide fragments formed from 2-CAP were chromatographically similar to those formed from hGH. The plasma kinetics of 2-CAP were compared to those of hGH with a RIA using polyclonal antiserum to hGH. After im and sc administration of 2-CAP (125 micrograms/kg), the area under the plasma concentration curve was 3.2- and 4.5-fold greater, respectively, than after administration of hGH (125 micrograms/kg). Both compounds had a greater area under the curve by the im than the sc route. 2-CAP had 2- to 3-fold greater bioavailability than hGH by the im and sc routes. Plasma from rats treated 30 min earlier with hGH im was immunoextracted and analyzed by Western blotting. A circulating immunoreactive fragment was detected which had similar electrophoretic mobility as a two-chain hGH product formed during the in vitro incubations of hGH with skeletal muscle and thyroid gland homogenates. The results indicate that hGH is proteolytically processed in peripheral tissue homogenates, with the formation of two-chain products. The greater bioavailability of 2-CAP suggests that metabolism of hGH to two-chain forms may influence the in vivo kinetics of hGH.
Amino Acid Sequence↗
Equilibrium denaturation of human growth hormone and its cysteine-modified forms.
The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.
Alkylation↗
Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers.
The composition and antibiotic permeability barrier of the outer membrane of Serratia marcescens were assessed in cells grown in vivo and in vitro. Intraperitoneal diffusion chambers implanted in rats were used for the in vivo cultivation of bacteria. Outer membranes isolated from log-phase bacterial cells recovered from these chambers were compared with membranes isolated from cells grown in vitro. Analysis revealed that the suspected 41-kilodalton porin and the OmpA protein were recovered on sodium dodecyl sulfate-polyacrylamide gels in equal quantities. Several high-molecular-weight proteins, thought to be iron starvation induced, appeared in the diffusion chamber-grown cells. The outer membrane permeability barriers to cephaloridine were similar in in vivo- and in vitro-grown cells based on permeability coefficient calculations. The permeability coefficient of cephaloridine in S. marcescens cells (30.3 x 10(-5) to 38.9 x 10(-5) cm s-1) was greater than that obtained for an Escherichia coli strain expressing only porin OmpC but smaller than those obtained for the E. coli wild type and a strain expressing only porin OmpF. Functional characterization of the suspected porin was performed by using the planar lipid bilayer technology. The sodium dodecyl sulfate-0.4 M NaCl-soluble porin from both in vitro- and in vivo-grown cells showed an average single-channel conductance in 1 M KCl of 1.6. A partial amino acid sequence (19 residues) was obtained for the S. marcescens porin. The sequence showed a very high homology to the E. coli OmpC porin. These data identified the S. marcescens outer membrane 41-kilodalton protein as a porin by both functional and amino acid analyses. Also, the methodology used allowed for efficient growth and recovery of diffusion chamber-grown bacterial cells and permitted identification of specific in vivo-induced changes in bacterial cell membrane composition.
Amino Acid Sequence↗
Liquid chromatographic control of the identity, purity and "potency" of biomolecules used as drugs.
The use of high-performance liquid chromatography (HPLC) in the control of rDNA-derived human insulin and human growth hormone is described. Powerful identity tests based upon reversed-phase HPLC separation of enzymatic digests have been developed. Size exclusion and reversed-phase assays are used to control higher molecular weight materials and monomeric derivatives, respectively, for both proteins. Finally, HPLC is used to control the relevant protein content, which in concert with other information controls the biopotency of the protein preparations.
Chromatography, High Pressure Liquid↗
Peptide and protein mapping by 252Cf-plasma desorption mass spectrometry.
The mapping of peptide digests by using fast atom bombardment mass spectrometry for evaluating the correctness of known or expected protein sequences is a well-established strategy. A similar approach ("PD mapping") is described which utilizes 252Cf-plasma desorption mass spectrometry (PDMS). This PD mapping approach is demonstrated and evaluated by screening the DNA-deduced sequences of recombinant interleukin-2 and human growth hormone. In the PD mapping experiment, the protein is cleaved either chemically or enzymatically and the molecular weights of the peptides predicted from the proposed amino acid sequence are compared with those determined mass spectrometrically. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptides through additional steps of enzymatic or chemical modification on the nitrocellulose-bound peptides. The PD mapping method is both fast and sensitive, requiring only low nanomole amounts per map.
Amino Acid Sequence↗
Secretion and folding of human growth hormone in Escherichia coli.
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Cloning, Molecular↗
Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone.
Two derivatives of biosynthetic human growth hormone, a sulfoxide and a mixture of two monodesamido isomers, have been isolated and characterized. The sulfoxide derivative arises from an oxidation of Met-14. The major site of deamidation is at Asn-149 with a minor site at Asn-152. In addition, a fraction has been isolated from a sample of human growth hormone that was maintained at 40 degrees C for 2 weeks. This fraction, the isolated impurities fraction, contains the sulfoxide and the desamido forms, thereby demonstrating that these derivatives are the primary degradation products of biosynthetic human growth hormone. The sulfoxide, the desamido, and the isolated impurities fraction exhibit full biological activity.
Amino Acid Sequence↗