scholarly journals Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

2003 ◽  
Vol 77 (14) ◽  
pp. 7804-7813 ◽  
Author(s):  
Wen Jun Liu ◽  
Hua Bo Chen ◽  
Alexander A. Khromykh
Keyword(s):  
Amino Acid ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Common Amino Acid

ABSTRACT A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

scholarly journals A Single Amino Acid Substitution in the West Nile Virus Nonstructural Protein NS2A Disables Its Ability To Inhibit Alpha/Beta Interferon Induction and Attenuates Virus Virulence in Mice

2006 ◽  
Vol 80 (5) ◽  
pp. 2396-2404 ◽  
Author(s):  
Wen Jun Liu ◽  
Xiang Ju Wang ◽  
David C. Clark ◽  
Mario Lobigs ◽  
Roy A. Hall ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Nonstructural Protein ◽  
Mutant Virus ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Wild Type ◽  
Alpha Beta ◽  
A30p Mutation

ABSTRACT Alpha/beta interferons (IFN-α/β) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-α/β responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNVKUN), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-β promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNVKUN genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-α/β in infected human A549 cells than that detected following wild-type WNVKUN infection. Consequently, replication of the WNVKUNNS2A/A30P mutant virus in these cells known to be high producers of IFN-α/β was abortive. In contrast, both the mutant and the wild-type WNVKUN produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-α/β production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-α/βγ receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNVKUN and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

scholarly journals Scanning Mutagenesis Studies Reveal a Potential Intramolecular Interaction within the C-Terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly and Secretion

2015 ◽  
Vol 89 (8) ◽  
pp. 4281-4295 ◽  
Author(s):  
Ren-Huang Wu ◽  
Ming-Han Tsai ◽  
Day-Yu Chao ◽  
Andrew Yueh
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Virus Assembly ◽  
Intramolecular Interaction ◽  
Rna Replication ◽  
The Other ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Virus Life Cycle

ABSTRACTThe NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transienttrans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated.IMPORTANCEThe reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

scholarly journals Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis

1996 ◽  
Vol 318 (3) ◽  
pp. 1041-1049 ◽  
Author(s):  
Himakshi PATEL ◽  
Jo BRAMALL ◽  
Helen WATERS ◽  
Maria C. DE BEER ◽  
Patricia WOO
Keyword(s):  
Amino Acid ◽  
Serum Amyloid A ◽  
Amyloid Fibrils ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Amyloid A ◽  
Amyloid Fibril Formation

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3–10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063–1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med.3, 387–407]. Wild-type rSAA protein was shown to form amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAAΔ1–11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.

scholarly journals Different molecular forms of rat kidney gp330, the dominant autoantigen of active Heymann nephritis

1995 ◽  
Vol 305 (3) ◽  
pp. 711-713 ◽  
Author(s):  
G G Jokhadze ◽  
A V Oleinikov ◽  
J J Kanalas ◽  
S P Makker
Keyword(s):  
Amino Acid ◽  
Direct Evidence ◽  
Rat Kidney ◽  
Single Amino Acid ◽  
Molecular Forms ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Cloning And Sequencing ◽  
Heymann Nephritis ◽  
Primary Structures

The primary structure, consisting of 1650 amino acid residues, of the C-terminal end of the dominant autoantigen of active Heymann Nephritis, gp330, from rat kidney was obtained by cloning and sequencing of cDNA clones. Comparison of this sequence with the previously published sequences of fragments of the C-terminal end of gp330 [Raychowdhury, Niles, McCluskey and Smith (1989) Science 244, 1163-1165] revealed certain differences in their primary structures. These differences included several single amino acid substitutions, replacement of a stretch of 15 amino acid residues by a different stretch of six amino acid residues, and different lengths of cytoplasmic domain (188 versus 213 amino acid residues). These findings of two different primary structures of gp330 provide direct evidence for the existence of two molecular forms of gp330.

scholarly journals Structural characterization of human aryl sulphotransferases

1999 ◽  
Vol 337 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Lulu A. BRIX ◽  
Ronald G. DUGGLEBY ◽  
Andrea GAEDIGK ◽  
Michael E. McMANUS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Substrate Specificities ◽  
Loop Region ◽  
Single Amino Acid Change

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

scholarly journals Identification of conformational neutralizing epitopes on the capsid protein of canine calicivirus

2001 ◽  
Vol 82 (7) ◽  
pp. 1695-1702 ◽  
Author(s):  
Yuichi Matsuura ◽  
Yukinobu Tohya ◽  
Masami Mochizuki ◽  
Kozo Takase ◽  
Takaaki Sugimura
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Conformational Epitopes ◽  
A Cell ◽  
Neutralizing Epitopes

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5′ and 3′ hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

scholarly journals Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

2001 ◽  
Vol 183 (19) ◽  
pp. 5743-5746 ◽  
Author(s):  
Babu S. Antharavally ◽  
Russell R. Poyner ◽  
Yaoping Zhang ◽  
Gary P. Roberts ◽  
Paul W. Ludden
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Paramagnetic Resonance ◽  
Specific Amino Acid ◽  
Dinitrogenase Reductase ◽  
Electron Paramagnetic ◽  
Hydrolysis Of

ABSTRACT Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

scholarly journals Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus

2005 ◽  
Vol 187 (5) ◽  
pp. 1552-1558 ◽  
Author(s):  
Masato Otsuka ◽  
Makoto Yasuda ◽  
Yuji Morita ◽  
Chie Otsuka ◽  
Tomofusa Tsuchiya ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ethidium Bromide ◽  
Vibrio Parahaemolyticus ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
Transmembrane Region ◽  
Mutant Proteins

ABSTRACT NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

scholarly journals The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

2006 ◽  
Vol 74 (10) ◽  
pp. 5595-5601 ◽  
Author(s):  
Cynthia L. Sears ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Augusto A. Franco
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

scholarly journals Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

1994 ◽  
Vol 304 (1) ◽  
pp. 289-293 ◽  
Author(s):  
T J Puranen ◽  
M H Poutanen ◽  
H E Peltoketo ◽  
P T Vihko ◽  
R K Vihko
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

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