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PubMed1979

Primary structure of a human IgA1 immunoglobulin. II. Isolation, composition, and amino acid sequence of the tryptic peptides of the whole alpha1 chain and its cyanogen bromide fragments.

As part of the strategy for determining the covalent structure of a human IgA1 molecule (Bur), a tryptic digest was prepared of the reduced and carboxymethylated alpha1 heavy chain. In addition to the main experiment, tryptic peptides were prepared from the succinylated aminoethylated alpha1 chain and from fragments obtained by CNBr scission of the alpha1 chain. Complete recovery of the peptides was impeded by the large size of some of the tryptic peptides and of the principal CNBr fragment, and difficulty in separating other glycopeptides. Twenty-eight tryptic peptides of the reduced and carboxymethylated alpha1 chain were purified and sequenced, accounting for more than 300 residues. Additional information was obtained by sequence analysis of trypudies described in this series of papers contributed to the complete sequence analysis of the alpha1 chain.

Amino Acid Sequence↗

PubMed1976

On the mechanism of malonyl-CoA-independent fatty-acid synthesis. Different properties of the mitochondrial chain elongation and enoylCoA reductase in various tissues.

1. NADPH-specific mitochondrial enoyl-CoA reductase can be assayed by a sensitive radioactive test, employing tritium-labelled NADPH, synthesized in a prefixed reaction from D-[1-3H]-glucose via the hexokinase and glucose-6-phosphate dehydrogenase reactions. 2. Liver, kidney cortex, heart muscle, skeletal muscle, brown adipose tissue, brain cortex, and aortic intimal tissue are investigated concerning chain lengths specificity of the chain elongation and the enoyl-CoA reductase. Medium-chain acyl-CoA compounds prove to be the best primers for the chain elongation. Enoyl-CoA reductases still show large incorporation rates with hexadecenoyl-CoA. 3. The differences in the chain lengths specificity of the chain elongation and enoyl-CoA reductase can be explained by the inhibitory effect of long-chain acyl-CoA derivatives on the 3-hydroxyacyl-CoA dehydrogenase. 4. The nucleotide specificity in the different tissues reveals two types of chain elongation: In addition to liver and kidney cortex, mitochondria of brown adipose tissue need NADH + NADPH for optimal chain elongation, whereas heart muscle, skeletal muscle and aortic intimal mitochondria only need NADH. 5. Different physiological roles are proposed for the two types. The "heart type" may be of importance in the conservation of reducing equivalents or acetate units in the anaerobic state, the "liver type" may play a role in the transfer of hydrogen from NADPH to the respiratory chain. In addition, the mitochondrial chain elongation may serve as bypass of the first part of the respiratory chain.

Adipose Tissue, Brown↗

PubMed1979

Cell-free synthesis and segregation of beta 2-microglobulin.

beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.

Amino Acid Sequence↗

PubMed1986

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s).

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.

Amino Acids↗

PubMed1982

A human-hybridoma system based on a fast-growing mutant of the ARH-77 plasma cell leukemia-derived line.

A rapidly-growing, HAT-sensitive cell line LICR-LON-HMy2 has been derived from the ARH-77 human plasma cell leukemia-derived line. It lacks the enzyme hypoxanthine phosphoribosyltransferase (EC 2.4.2.8). Hybrids have been reproducibly made for more than a year and in independent laboratories with lymphocytes from tonsils, lymph nodes and peripheral blood and tonsil lymphocytes cultured with antigens. The parent line and hybrids are very robust in culture and double in 20-30 h. Hybrids clone easily and have stable karyotypes, most with modal numbers in the sixties. Stable Ig secretion patterns have been observed over 20-30 passages, and after cloning. It was estimated that about half the hybrids produce new immunoglobulin chains in addition to the parent cell line's IgG1 (kappa light chain). High, but not limiting, density hybridoma cultures (approximately 5 x 10(5) cells/ml) typically produce 0.25-2 micrograms/ml immunoglobulin per day, but some hybrids produce more. A high-secreting variant of LICR-LON-HMy2 has been derived. The LICR-LON-HMy2 line is human and is distinct from the HAT-sensitive human B cell-lines SKO-007 and GM 1500-6TG Al 2. It is available for distribution.

Antibodies, Monoclonal↗

PubMed1996

The vaginal introitus: a novel site for Chlamydia trachomatis testing in women.

OBJECTIVE: We evaluated the vaginal introitus as a noninvasive sampling site for testing for Chlamydia trachomatis. STUDY DESIGN: Swabs from the vaginal introitus were obtained from 300 women attending a sexually transmitted diseases clinic and tested for the presence of Chlamydia trachomatis by polymerase chain reaction. Additionally, 200 of these women self-collected an additional introitus swab and submitted a urine sample for polymerase chain reaction testing. These samples were compared with polymerase chain reaction, culture, and enzyme immunoassay for Chlamydia trachomatis from endocervical samples and polymerase chain reaction and culture on urethral swabs. Patients were determined to be infected with Chlamydia trachomatis by a positive culture result from any site or a confirmed positive result by polymerase chain reaction with an alternate primer. RESULTS: The sensitivity of vaginal introitus swabs obtained by health care providers for the detection of urogenital Chlamydia trachomatis was 92% (95% confidence interval 83 to 100), greater than polymerase chain reaction, culture, or enzyme immunoassay of the cervix or urethra. The sensitivity by polymerase chain reaction of patient self-collected swabs was 81%. Sampling of the vaginal introitus, by both health care workers and the patient herself, performed as well as commonly used diagnostic tests that require vaginal speculum examination. The sensitivity of polymerase chain reaction testing of urine samples was 73%. CONCLUSION: The vaginal introitus represents a highly effective noninvasive specimen collection site for Chlamydia trachomatis testing by polymerase chain reaction. Self-collection of introitus specimens may revolutionize sexually transmitted disease testing by eliminating the need for a speculum examination by skilled health care personnel.

Chlamydia Infections↗

PubMed2006

Chemical synthesis and molecular recognition of phosphatase-resistant analogues of phosphatidylinositol-3-phosphate.

The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. To provide chemical tools to study the changes in cell physiology mediated by these lipids, three new metabolically stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P) were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-(monofluoromethyl)phosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl, and dibutyryl chains. In addition, we introduce a new phosphorylation reagent, (monofluoromethyl)phosphonyl chloride, which has general applications for the preparation of "pKa-matched" monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labeled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)P analogues into membrane-mimetic dodecylphosphocholine micelles. In addition, the PtdIns(3)P analogues with dioleoyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis.

Humans↗

PubMed2004

Functional and surface-active membranes from poly(vinylidene fluoride)-graft-poly(acrylic acid) prepared via RAFT-mediated graft copolymerization.

Poly (vinylidene fluoride) (PVDF) with "living" poly (acrylic acid) (PAAc) side chains (PVDF-g-PAAc) was prepared by reversible addition-fragmentation chain transfer (RAFT)-mediated graft copolymerization of acrylic acid (AAc) with the ozone-pretreated PVDF. The chemical composition and structure of the copolymers were characterized by elemental analysis, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The copolymer could be readily cast into pH-sensitive microfiltration (MF) membranes with enriched living PAAc graft chains on the surface (including the pore surfaces) by phase inversion in an aqueous medium. The surface composition of the membranes was determined by X-ray photoelectron spectroscopy. The morphology of the membranes was characterized by scanning electron microscopy. The pore size distribution of the membranes was found to be much more uniform than that of the corresponding membranes cast from PVDF-g-PAAc prepared by the "conventional" free-radical graft copolymerization process. Most important of all, the MF membranes with surface-tethered PAAc macro chain transfer agents, or the living membrane surfaces, could be further functionalized via surface-initiated block copolymerization with N-isopropylacrylamide (NIPAAM) to obtain the PVDF-g-PAAc-b-PNIPAAM MF membranes, which exhibited both pH- and temperature-dependent permeability to aqueous media.

Acrylic Resins↗

PubMed1994

Characterization of the antigens recognized by two monoclonal antibodies reactive with basal-layer keratinocytes of human epidermis.

Two monoclonal antibodies, GR3 and GR4, reactive with the basal-layer keratinocytes of human epidermis, were derived by immunization of Balb/c mice with glycoproteins isolated from cultured keratinocytes by lectin-affinity chromatography. Immunoprecipitation of Triton X-100 extracts from human keratinocytes metabolically labelled with D-[1-14C]glucosamine revealed that GR3 recognized a major glycoprotein with migration properties identical with those of a glycoprotein (reduced form M(r) 126,000) which was previously shown to be implicated in intercellular adhesion [Roberts and Brunt (1985) Biochem J. 232, 67-70]. In their unreduced forms the antigens recognized by GR3 and GR4 both migrated as two bands with M(r) values of 118,000 and 147,000. Comparison of 125I-labelled glycoproteins immunoprecipitated by GR3, GR4 and integrin antibodies revealed that, under reducing conditions, the major band immunoprecipitated by both GR3 and GR4 co-migrated with the alpha 3 and beta 1 integrin chains. In addition the immunoprecipitate obtained with GR4 contained an additional band co-migrating with the alpha 2 integrin chain. Sequential immunoprecipitation studies with GR3, GR4 and integrin antibodies confirmed that GR3 is directed against the alpha 3 integrin chain, whereas GR4 is directed against the beta 1 chain. These studies also indicate that some of the alpha 2 integrin chains on keratinocytes may be associated with a beta-chain not recognized by the antisera against the beta 1 integrin chain used in this study.

Animals↗

PubMed1985

Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation.

The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1992

Carbodiimide-mediated coupling of benzenepentacarboxylate to human hemoglobin: structural and functional consequences.

We have examined the covalent modification of HbA with BPC (benzenepentacarboxylate) whose carboxyl groups were activated with EDC [1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide]. Reaction of deoxy-HbA at pH 8 with a 10-fold excess of BPC, preactivated with a 2-fold excess of EDC for 5 minutes, followed by anion-exchange chromatography, gives three components with p50 values of 1.15 (unreacted HbA), 11.7 and 7.6 mm of Hg at 20 degrees C (50 mM Bis-Tris pH 7.0). Component III does not dissociate into dimers upon dilution, but components I and II do. When deoxy-HbA is reacted at pH 6 with 10-fold BPC, preactivated with two-fold EDC for 5 minutes, the resultant HbA derivatives can be separated into three components, with p50 values at pH 7 of 14.2, 10.2 and 5.2 mm of Hg, respectively. All three components are stable tetramers. Oxygen binding by all of the covalent HbA-(BPC)x complexes is cooperative, pH sensitive, but IHP insensitive. The latter observation suggest that BPC is covalently bound to HbA's DPG/IHP binding site. This conclusion is corroborated by reversed phase HPLC analysis which shows that all five modified HbAs contain at least one modified beta chain. In addition, 4 of the 5 derivatives also contain modified alpha chains. No inter or intratetramer crosslinks are observed.

Benzene Derivatives↗

PubMed1999

Differential tissular expression and localization of type IV collagen alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains and their mRNA in normal breast and in benign and malignant breast tumors.

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha chains. We investigated the cellular regulation and origin of these alpha(IV) chains in normal and neoplastic breast tissues by immunohistochemistry by using alpha(IV) chain-specific antibodies and by in situ hybridization. In normal breast, alpha1(IV) and alpha2(IV) chains were stained in all BM, whereas alpha5(IV) and alpha6(IV) chains were restrictively localized in a linear pattern in the BM of the mammary gland. Similar immunostaining profiles were observed in benign breast tumors and in the intraductal components of invasive ductal carcinoma. However, in invasive ductal carcinoma, alpha1(IV) and alpha2(1V) chains were discontinuously or negatively stained in the cancer cell nests, and the assembly of alpha5(IV) and alpha6(IV) chains into the BM was completely inhibited. Coexpression of alpha5(IV) and alpha6(IV) chains was related to the localization of alpha-smooth muscle actin (alpha-SMA)-positive myoepithelial cells. By in situ hybridization, in fibroadenoma and invasive ductal carcinoma, the signals for alpha1(IV) and alpha2(IV) mRNA were abundant in stromal cells. However, the signals for alpha5(IV) and alpha6(IV) mRNA were not seen in any of these cells. In contrast, in intraductal papilloma, coexpression of alpha1 (IV)/alpha2(IV) mRNA and alpha5(IV)/alpha6(IV) mRNA was identified in epithelial cells. The results indicate that the mammary gland forms a second network of BM composed of alpha5(IV)/alpha6(IV) chains, in addition to the classic network of alpha1(IV)/alpha2(IV) chains. The expression of type IV collagen alpha chains seems to be differentially regulated by the epithelial-myoepithelial interaction and to be associated with the invasive potential of breast cancer.

Breast↗

PubMedDate not supplied

["Oxygen regulation" of the respiratory chain composition in the yeast Debaryomyces hansenii under multiple stress].

It was shown that two stress factors, hypoxia and hyperosmotic shock, if applied simultaneously to the yeast Debaryomyces hansenii, display an antagonistic mode of interaction, which results in an increased degree of halophily of this microorganism under microaerobic conditions. Studies of the effects of respiration inhibitors (sodium azide and salicyl hydroxamic acid, SHA) and of the pattern of changes in the composition of the respiratory chain of Debaryomyces hansenii under the stated stress conditions led to the suggestion of three (or four) chains of electron transfer functioning simultaneously in the cell: the classical respiratory chain involving cytochrome-c oxidase, an alternative respiratory chain involving a cyanide- and azide-resistant oxidase, and additional respiratory chains involving oxidases resistant to salt, azide and SHA. Thus, the antagonistic mode of interaction between hypoxia and hyperosmotic shock results from the redirection of the electron flow from the salt-susceptible respiratory systems to the salt-unsusceptible ones encoded by "the hypoxia genes" and activated (induced) under microaerobic conditions.

Anaerobiosis↗

PubMed1987

The laminin B2 chain has a multidomain structure homologous to the B1 chain.

Laminin (Mr = 850,000) is a large basement membrane-specific glycoprotein composed of three chains: A, B1, and B2. Previously, we have reported the primary structure of the B1 chain of mouse laminin deduced from sequencing cDNA clones (Sasaki M., Kato, S., Kohno, K., Martin, G. R., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 935-939). Here we report the isolation of overlapping cDNA clones spanning 7642 bases which encode the entire B2 chain. The nucleotide sequence of the clones contains an open reading frame of 4821 bases coding for a protein of 1607 amino acids including 33 amino acids of a presumptive signal peptide. The mRNA for the B2 chain contains 2.5 kilobases of 3'-untranslated region. The deduced amino acid sequence indicates that the B2 chain consists of six distinct domains, including two domains with alpha-helical, coiled-coil structures, two domains with cysteine-rich homologous repeats, and two globular domains. These structural features of the B2 chain are similar to those of the B1 chain. In addition, the amino acid sequences of the B2 and B1 chains demonstrate considerable homology, suggesting that the genes for these two chains arose from a common ancestor.

Amino Acid Sequence↗

PubMed1993

Molecular cloning of rat calpactin I heavy-chain cDNA whose expression is induced in v-src-transformed rat culture cell lines.

A cDNA clone, termed N02, was isolated from a rat 3Y1 cDNA library using a differential screening procedure. The amount of N02 mRNA was increased in 3Y1 cells transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-3Y1) compared with that in 3Y1 cells. Sequence analysis revealed that N02 cDNA encodes a p36 calpactin I heavy chain. In addition, the amount of a p11 calpactin I light-chain mRNA was also increased in v-src-transformed rat culture cells. The expression of both p36 and p11 genes was also induced in several sets of transformed cells, including v-H-ras, v-mos- or SV40-transformed 3Y1 cells. As the expression of p36 and p11 genes was increased in transformed cells irrespective of the nature of oncogene products, overexpression of p36 and p11 genes might be involved in the key step of cellular transformation.

Amino Acid Sequence↗

PubMed1985

Genetic complexity and expression of human class II histocompatibility antigens.

The genes encoding nearly all of the serologically defined class II antigens of the major histocompatibility complex have been isolated. Three class II loci have been studied in great detail. The DR region contains a single alpha gene and 3 beta chain genes, 1 of which is a pseudogene. The DR alpha chain gene has been linked to a DR beta gene which encodes a beta protein which contains the serological determinant MT3. A second cosmid cluster contains 2 beta genes, 1 of which encodes the DR4 allospecificity. The identification of these genes has been made by the comparison of amino terminal sequences of DR molecules obtained from a DR4 cell line and the deduced protein sequences of the beta 1 exons from cosmid and phage clones. A conserved element including the promoter and signal sequence is found at the 5' end of each of the 3 DR beta genes. Additionally, this element occurs three more times in the DR region, raising the question of whether additional beta chain genes might be found. The DQ region contains 2 pairs of genes, 1 of which encodes the DQ antigen. The 2nd pair of genes, called DX alpha and beta, appears to be capable of expressing a DQ-related product, although, to date, there is no evidence for its expression. The DP region also contains 2 pairs of genes. One pair encodes the DP antigen while the 2nd alpha-beta pair is shown to be composed of pseudogenes. The location of polymorphic regions in these genes and aspects of their relationship to the serology, evolution, and function of the class II MHC are discussed. The control of expression of class II genes by gamma-interferon has been examined. The promoters of class II genes are characterized by two conserved sequences common to all alpha and beta chain genes as well as by conserved sequences specific for either alpha or beta chain genes. In addition to studies of expression by DNA-mediated gene transformation, a system for the gene transfer of MHC antigens utilizing transmissible retrovirus vectors is described. Retrovirus vectors have been used to transmit DR alpha, DR beta, and the invariant chain (gamma) sequences to recipient cells with resultant expression of these proteins.

Alleles↗

PubMed1993

A novel quinoprotein methanol dehydrogenase containing an additional 32-kilodalton peptide purified from Acetobacter methanolicus: identification of the peptide as a MoxJ product.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain in addition to an ethanol oxidase respiratory chain. In this study, two different forms of methanol dehydrogenase (type I and II MDHs) were purified from A. methanolicus grown on methanol. Type I MDH was more basic (pI of 8.0) and smaller (M(r) of 148K) than type II MDH (pI of 6.7 and M(r) of 177K). Type I MDH consisted of alpha and beta subunits of 62 and 10 kDa, which has the same alpha 2 beta 2 conformation as the enzymes purified so far. The type II MDH contained an additional peptide of 32 kDa, of which a single copy was estimated to bind to the alpha 2 beta 2-MDH judging from the whole molecular weight and the stoichiometry of each subunit determined in sodium dodecyl sulfate-high-performance gel filtration chromatography. Compared with type I MDH, type II MDH exhibited a lower enzyme activity, but the electron-transfer activity to cytochrome cL was much more resistant to the inhibition with NaCl or EDTA. The possibility could be excluded that type II MDH is an artificial complex of type I MDH with a 32-kDa peptide, since it was inducible with methanol and could be detected in the periplasm, as well as the other subunits. Furthermore, the N-terminal amino acid sequence of the 32-kDa peptide showed a high homology to that of the moxJ product deduced from the DNA sequence of Paracoccus denitrificans or Methylobacterium extorquens AM1.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetobacter↗

PubMed1980

The arginines of cytochrome c. The reduction-binding site for 2,3-butanedione and ascorbate.

The reactions of both ferric and ferrous horse heart cytochrome c with 2,3-butanedione, pH 7.5, 0.05 M bicarbonate buffer, under a variety of solution conditions, have been examined using amino acid integrity as the primary criterion. The only observable structural alteration was the modification of both the arginyl side chains. The reaction profiles were found to be dependent upon the presence or absence of borate and ascorbate. The single-phased modification of the arginyl side chains in ferrocytochrome c in the absence of borate is rendered biphasic in the presence of borate (borate/reagent ratio of 0.04) through substantial lowering of the rate of reaction of one of the two arginyl side chains. The addition of ascorbate inhibits the reaction in a competitive manner, particularly of the arginyl side chain undergoing rapid modification in its absence. The reactivity of both arginyl side chains to reagent for the ferricytochrome was appreciably larger than for ferrocytochrome c. The addition of reagent to ferricytochrome c also reduces heme iron, which is discerned from the development of a native-like spectrum in the region 500 to 600 nm. The differences in the reactivities of the arginyl side chains to 2,3-butanedione in the two valence states of heme iron are the reflection of small, but definite, conformational differences at the two sites in the two valence states of the protein. The concurrent reduction of heme iron and the modification of the arginyl side chains localizes the reduction and the reaction site for 2,3-butanedione. The inhibition by ascorbate of the reaction of one of the two arginines also identifies the binding domain. Of the two arginines, Arg-38 is suggested to be the ascorbate-binding site.

Animals↗