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1.
N Goshima  Y Kano  H Tanaka  H Tanaka  K Kohno  K Yasuzawa  F Imamoto 《Gene》1992,121(1):121-126
Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.  相似文献   

2.
Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64----Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40-350-fold. The His-64----Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were approximately 50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were approximately 5-15-fold higher than those for the native protein. All mutants studied (except His-64----Tyr) showed approximately 10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O2 and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O2 affinities, and 50-380-fold greater M (KCO/KO2) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.  相似文献   

3.
Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.  相似文献   

4.
Domain II mutants of Pseudomonas exotoxin deficient in translocation   总被引:16,自引:0,他引:16  
Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.  相似文献   

5.
An efficient random mutagenesis procedure coupled to a replica plate screen facilitated the isolation of mutant subtilisins from Bacillus amyloliquefaciens that had altered autolytic stability under alkaline conditions. Out of about 4000 clones screened, approximately 70 produced subtilisins with reduced stability (negatives). Two clones produced a more stable subtilisin (positives) and were identified as having a single mutation, either Ile107Val or Lys213Arg (the wild-type amino acid is followed by the codon position and the mutant amino acid). One of the negative mutants, Met50Val, was at a site where other homologous subtilisins contained a Phe. When the Met50Phe mutation was introduced into the B. amyloliquefaciens gene, the mutant subtilisin was more alkaline stable. The double mutant (Ile107Val/Lys213Arg) was more stable than the isolated single mutant parents. The triple mutant (Met50Phe/Ile107Val/Lys213Arg) was even more stable than Ile107Val/Lys213Arg (up to two times the autolytic half-time of wild-type at pH 12). These studies demonstrate the feasibility for improving the alkaline stability of proteins by random mutagenesis and identifying potential sites where substitutions from homologous proteins can improve alkaline stability.  相似文献   

6.
Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides (1). The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues #24-45. An inhibitory region for PDE alpha/beta is within residues #80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, mutants were synthesized and utilized in PDE inhibition assays. The following mutants exhibited a decreased ability to inhibit PDE alpha/beta: Tyr84----Gly; Arg24----Gly; and Arg33----Pro. Sequence comparisons with cone PDE gamma indicate that there is identity within these functional regions.  相似文献   

7.
The single coding exons of the cloned genes encoding two human arylamine N-acetyltransferases (NAT1 and NAT2) were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based phagemid vector pKEN2 for production of the recombinant proteins in Escherichia coli strain XA90. Induction of cultures grown from selected bacterial transformants resulted in the production of substantial quantities of soluble recombinant human NAT1 and NAT2 with identical electrophoretic, immunologic and catalytic properties to those expressed in mammalian cell culture or in human liver. Oligonucleotide-directed mutagenesis of recombinant human NAT2 was then employed to determine the relative importance of 3 highly conserved cysteine residues in the enzyme's catalytic mechanism. Substitution of cysteine with glycine at position 68 of the 290 amino acid protein molecule (Cys68----Gly) resulted in the production of normal quantities of immunoreactive NAT2 which was completely devoid of enzyme activity, suggesting that the sulfhydryl group of Cys68 is directly involved in the transfer of acetate from the essential cofactor CoASAc to acceptor amine substrates. On the other hand, the mutations producing Cys44----Gly and Cys223----Gly led to the production of enzymatically active NAT2 proteins with markedly reduced in vitro stability, suggesting that substitution of either of these amino acids may cause alterations in the tertiary structure of the native enzyme.  相似文献   

8.
RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The Km of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.  相似文献   

9.
Accessible surfaces of the HSA molecule in N-, F- and B-forms were studied in the present work by tritium labelling method which allowed to obtain detailed information on N-F- and N-B-transitions. In was shown that the F-form in comparison top the N-form is characterized by more high accessibility of Ser, Ala, Ile, Tyr, Phe, His, Arg, Pro, Val and Phe residues and in the B-form Tyr, Ser, Arg, Gly, Ile, Phe and Pro residues turn to be highly accessible. Full accessible surfaces of protein molecule at N-F- and N-B-transitions increase respectively from 39,000 to 70,400 A2 and from 39,000 to 47,000 A2. Basing on the prevailing increase of hydrophobic residues accessibility it is supposed that the molecule expansion testifies the separation of the subunits forming the molecule.  相似文献   

10.
A family of mutant amidases has been derived by experimental evolution of the aliphatic amidase of Pseudomonas aeruginosa strain PAC1. Mutation amiE16, in the structural gene for the enzyme, results in the production of the mutant B amidase by strain B6. This strain, unlike the wild-type, can utilize butyramide for growth. Strain B6 gave rise by a single mutational event to strain V9, utilizing valeramide, and strain PhB3, utilizing phenylacetamide. Strain V9 was not itself able to utilize phenylacetamide but gave rise by mutation to the phenylacetamide-utilizing mutant PhV1. Peptide 108 was isolated from chymotryptic digests of mutant amidases from strains B6, PhB3 and PhV1, but could not be detected in chymotryptic digests of the wild-type amidase. The sequence of peptide 108 was established as Met-Arg-His-Gly-Asp-Ile-Phe. Thermolytic digests of mutant amidases from strains B6, PhB3, PhV1 and V9 were compared with digests of the wild-type amidase. A peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val was found in the digest of the wild-type amidase and was replaced in the digests of the mutant amidases by a peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val, Phe. Mutation amiE16 is common to the four mutant enzymes and can be accounted for by the mutation Ser leads to Phe. The sequence of the chymotryptic peptide corresponds with the N-terminal sequence of the amidase protein, and can also be related to the thermolysin peptides. It is concluded that mutation amiE16 is a Ser leads to Phe change at position 7 from the N-terminus and the effect of this on the enzyme conformation is discussed.  相似文献   

11.
M Wada  K Kutsukake  T Komano  F Imamoto  Y Kano 《Gene》1989,76(2):345-352
The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that DNA inversion mediated by hin, gin, pin and rci but not by cin is blocked in a hupA hupB double mutant, although inversions in these systems occur in the hupA or hupB single mutant as efficiently as in the wild-type strain. These findings show that HU protein participates in site-specific DNA inversion in E. coli and that only one subunit, either HU-1 or HU-2, is sufficient for this inversion.  相似文献   

12.
The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases contain a domain consisting of three contiguous copies of the fibronectin (FN)-derived type II homology unit (T2HU), T2HU-1, T2HU-2, and T2HU-3. To investigate the functional role of this domain, we have constructed plasmids expressing beta-galactosidase fusion proteins with one or more of the CLG4B-derived T2HU. The gelatin binding assays demonstrate that a single copy of T2HU-2 renders beta-galactosidase capable of binding gelatin. The three repeats, however, differ dramatically in their capacity to bind gelatin, with T2HU-1 and T2HU-3 having significantly less binding activity than T2HU-2. Using alanine scanning mutagenesis we have defined the amino acid residues (Arg307, Asp309, Asn319, Tyr320, Asp323) that are critical for gelatin binding of T2HU-2. The low gelatin binding of T2HU-1 compared to T2HU-2 was traced to the non-conserved residues Ala228-Ala and Leu253-Pro. The results suggest that the gelatin binding of the type IV collagenase proenzyme is mediated by the FN-like domain, although the presence of another gelatin-binding site cannot be excluded. The FN domain-mediated binding, however, is not a rate-limiting step in the hydrolysis of gelatin by the enzyme.  相似文献   

13.
The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.  相似文献   

14.
In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.  相似文献   

15.
Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys40 or Arg143 accounts for the human enzyme's marked preference for Phe8 over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys40 or Arg143 to neutral residues. Lys40 was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-chymase 1. The Lys40Ala mutant is a dog-like enzyme retaining strong preference for Phe8 but with Tyr4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg143Gln mutant, contrary to prediction, remains highly Phe8-selective. Therefore, Lys40, but not Arg143, contributes to human chymase's remarkable preference for AT II generation over destruction.  相似文献   

16.
Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.  相似文献   

17.
Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides. The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues # 24-45 and an inhibitory region for PDE alpha/beta within residues # 80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, peptides of PDE gamma # 63-87 and mutant peptides were synthesized and utilized in PDE inhibition assays. The following peptides exhibited a decreased ability to inhibit PDE alpha/beta: All were from PDE gamma # 63-87; PDE gamma Tyr 84----Gly, PDE gamma Phe 73----Gly and PDE gamma Gln 83----Gly.  相似文献   

18.
To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct energy transfer from the B800-850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.  相似文献   

19.
Mucopolysaccharidosis type II (MPS II) is a rare X-linked disorder caused by alterations in the iduronate-2-sulfatase (IDS) gene. In this study, IDS activity in peripheral mononuclear blood monocytes (PMBCs) was measured with a fluorimetric enzyme assay. Urinary glycosaminoglycans (GAGs) were quantified using a colorimetric assay. All IDS exons and intronic flanks were bidirectionally sequenced. A total of 15 mutations (all exonic region) were found in 17 MPS II patients. In this cohort of MPS II patients, all alterations in the IDS gene were caused by point nucleotide substitutions or small deletions. Mutations p.Arg88His and p.Arg172* occurred twice. All mu- tations were inherited except for p.Gly489Alafs*7, a germline mutation. We found four new mutations (p.Ser142Phe, p.Arg233Gly, p.Glu430*, and p.Ile360Tyrfs*31). In Epstein-Barr virus (EBV)-immortalized PMBCs derived from the MPS II patients, no IDS protein was detected in case of the p.Ser142Phe and p.Ile360Tyrfs*31 mutants. For p.Arg233Gly and p.Glu430*, we observed a residual expression of IDS. The p.Arg233Gly and p.Glu430* mutants had a residuary enzymatic activity that was lowered by 14.3 and 76-fold, respectively, compared with healthy controls. This observation may help explain the mild disease phenotype in MPS II patients who had these two mutations whereas the p.Ser142Phe and p.Ile360Tyrfs*31 mutations caused the severe disease manifestation.  相似文献   

20.
By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.  相似文献   

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