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Biomedical subjects

I C Kim

Publications and source records attributed to I C Kim.

At least 55 records · Page 3Linked to original sources
PubMed1987

Radioimmunoassay for testicular cytochrome c (ct). Evidence for the presence of apocytochrome ct pool in rat testis extract.

Mammalian testis contains a tissue-specific testicular cytochrome c (cyt ct). By immunizing rabbits with rat cyt ct and phosphorylated albumin (pBSA), rabbit anti-cyt ct was produced. Then the antiserum was applied to phosphorylated bovine serum albumin and rat somatic cyt c (cs)-Sepharose affinity columns to remove cross-reacting antibodies. The resultant anti-cyt ct was highly specific for cyt ct. From immunoblot assays, no protein other than cyt ct in rat testis extract was bound by the anti-cyt ct. By using the anti-cyt ct, radioimmunoassay (RIA) was developed for the quantitation of cyt ct in rat testis extract. The observation that the RIA did not bind rat cyt cs (1-1000 pmol), and other rat tissue extracts (kidney, heart, lung) further indicated that the RIA was highly specific for rat cyt ct. Separately, the concentration of holocyt ct was determined using CM-cellulose chromatography and subsequent spectral analysis on the same testis extract. The total cyt ct concentration in the rat testis extract determined by the RIA was about 3-fold higher than those determined by the latter techniques. Since the affinity purified anti-horse cyt c cross-reacted with both horse holo- and apocyt c, anti-rat cyt ct will cross-react with rat apocyt ct. Thus the concentration of cyt ct quantitated by the polyclonal anti-cyt ct-based RIA probably included apocyt ct concentration as well. Therefore, the higher cyt ct concentration determined by the RIA was probably attributed to the presence of the apocyt ct in the testis extract. The presence of the high concentration of the apocyt ct pool in testis is probably necessary to maintain continuous spermatogenesis, during which holocyt ct is incorporated into sperm mitochondria.

Animals↗
PubMed1987

Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells. A 92-kDa Glycoprotein.

The structure of the lutropin/choriogonadotropin (LH/CG) receptor has been studied by immunoprecipitating the receptor from biosynthetically labeled cultured Leydig tumor cells (designated MA-10). This was performed by binding human choriogonadotropin (hCG) to the labeled cells, solubilizing the hormone-receptor complex, partially purifying the complex by lectin chromatography, and immunoprecipitating the complex with an antibody that recognizes receptor-bound hCG. The conditions used for the release of the radiolabeled receptor from the immunoprecipitate and the subsequent analysis of this material on sodium dodecyl sulfate gels allowed us to determine directly the structure of the free (not hormone-occupied) LH/CG receptor. From experiments using cells labeled with [35S]methionine and [35S]cysteine, we show that the LH/CG receptor is composed of a single polypeptide chain that migrates as a 92-kDa protein on sodium dodecyl sulfate gels whether analyzed in the absence or presence of reducing agents. Other studies presented demonstrate that the LH/CG receptor is a glycoprotein.

Cell Line↗
PubMed1987

Antigenic analysis of human trophoblast membrane: detection of a lymphocyte cross-reactive antigen.

The antigenicity of human syncytiotrophoblast membrane (TM) was analyzed using rabbit antiserum raised against TM. From immunoblot analysis, about ten protein bands in TM were recognized by the anti-TM. These included placental alkaline phosphatase and TM-bound albumin. From limited antigenic specificity studies, these antigens, with the exception of albumin, were not detectable in membranes of liver, kidney, heart, and erythrocyte, or in human normal serum. Therefore, these antigens appear to be placental membrane specific. Analysis of lymphocyte membrane by immunoelectrophoresis, crossed immunoelectrophoresis, and immunoblot techniques revealed that a single protein band, designated "40 kDa," was cross-reactive with the anti-TM antiserum, indicating that this antigen is shared commonly between placenta and lymphocyte membranes. Experimental evidence suggesting that the 40-kDa membrane antigen is probably a unique trophoblast-lymphocyte cross-reactive antigen is based on the following observations: the 40-kDa antigen was not detectable in liver, heart, and kidney membrane; beta 2-microglobulin (12,000 daltons) was not detectable in our TM preparation; and the lymphocyte membrane showed only a single protein band at 40,000 daltons, not at 12,000 for beta 2-microglobulin.

Antigens, Surface↗
PubMed1987

Effects on fertility of rabbits after active immunization with rat testicular cytochrome ct.

A Male rabbit was immunized with rat testicular cytochrome (Cyt) ct and mated with normal, unimmunized females. The matings resulted in abnormal pregnancies: no offspring or stillborn or undersized liveborn offspring weighing 25-30 gm each. Another unusual observation was that fur-pulling behavior, normally exhibited by pregnant female rabbits at the end of the gestational period, was absent in all of these pregnancies. Therefore, immunization of a normal rabbit with testicular cyt ct appeared to interfere with physiological and behavioral aspects of pregnancy in normal female rabbits. The immunological basis of these findings remains to be determined.

Animals↗
PubMed1987

Characterization of monoclonal antibodies to cytochrome c: analysis of the antigenic structure of holo- and apo-cytochromes, and CNBr-peptide fragments of horse cytochrome c.

Five mouse hybridoma cell lines secreting SA, SB, SC, SD, and SE monoclonal antibodies (McAb) to cytochrome c have been produced. From the cross-reactivities of these McAb with various vertebrate cytochromes c, the antigenic sites for SA and SB McAb were proposed to be at Thr(89)-Glu(92)-Ala(96) and Asn(103), respectively. The binding site for other McAb have not been determined. Cross-reactivity studies based on enzyme-linked immunosorbent assays and dot immunobinding assays indicated that SA, SB, and SC McAb did not bind to apo-cytochrome c nor to any of the three CNBr-peptide fragments. This observation suggests that (i) the antigenic specificity of these McAb is dependent on the conformatiuon of the antigenic site which is inherent to the native holoprotein molecule and (ii) the ordered conformation in the C-terminal regions of holo-cytochrome c is destroyed during CNBr-peptide fragmentation. On the other hand, the lack of binding of SD and SE McAb to apo-cytochrome c indicates that these McAb are also specific for conformational sites. The binding of SD and SE McAb to the heme-containing A-peptide fragment (residues 1-65) suggests that the conformation around the heme, as possible antigenic sites, are stable because of the thioether linkages by the Cys residues.

Animals↗
PubMed1986

Antigenic and size differences between somatic and testicular cytochromes c.

Enzyme-linked immunosorbent assays (ELISA) of somatic (cs) and testicular (ct) cytochromes of rat, mouse, rabbit, and beef with rabbit anti-rat cyt ct antibody exhibited two different antigenic profiles, indicating the presence of two different antigenic structure between cyt ct and cs. SDS-polyacrylamide gel electrophoresis of cyt c showed that the molecular size of rat, rabbit and beef cyt ct is slightly smaller than that of their cyt cs. However, the electrophoretic mobility of mouse cyt ct is almost identical to that of mouse cyt cs, but slightly slower than that of rat cyt ct. These results indicate that mouse and rat cyt ct are different despite the identical amino acid sequences for both rat and mouse cyt cs.

Animals↗
PubMed1986

Antigenic analysis of testicular cytochromes c using monoclonal antibodies.

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.

Animals↗
PubMed1984

Comparative in vitro spermicidal effects of (+/-)-gossypol, (+)-gossypol, (-)-gossypol and gossypolone.

The comparative in vitro spermicidal effects of (+)-gossypol, (-)-gossypol and (+/-)-gossypol were evaluated on the spermatozoa of human, monkey, rabbit, mouse, rat and hamster. The spermicidal effects of gossypol isomers were also compared with those of gossypolone, which is a proposed major metabolite of gossypol. Gossypol isomers and gossypolone were all spermicidal. (+)- and (-)-Gossypol demonstrated spermicidal activities at the same concentration at which (+/-)-gossypol shows spermicidal effects on the spermatozoa of all species tested. However, gossypolone was less potent than the gossypol isomers. The spermicidal action of gossypol may be a nonspecific effect unrelated to the antifertility mechanism of orally administered gossypol, since (+)-gossypol which is not an effective male antifertility agent also showed the equivalent spermicidal effect to that of (+/-)-gossypol.

Animals↗
PubMed1982

Immunological characterization of cytochrome H-450 in rat tissues.

Rabbit antiserum produced against rat liver cytochrome H-450 was specific for cytochrome H-450. The antiserum did not react with hemolysate, microsomal and mitochondrial fractions of liver, and tissue extracts from heart, lung skeletal muscle, and testis of rat. With the monospecific antiserum, a rocket immunoelectrophoretic assay method was developed for the quantitation of the antigen with a sensitivity of 25 ng. By using rocket immunoelectrophoresis, the total amounts of the antigen found in liver, kidney, and brain of 20 rats were 33.6, 3.6, and 1.3 mg, respectively. It appears that the antigens in liver, kidney, and brain are immunologically identical. From immunological studies with subcellular fractions of rat liver, the antigen was found only in the postmicrosomal fraction. This indicates that the antigen is not a precursor or a proteolytic product of known cytochromes in mitochondria or microsomes. Therefore, cytochrome H-450 is a unique cytosolic protein found in brain, kidney, and liver.

Animals↗
PubMed1981

Immunological characterization of cytochrome b9 in crude extracts from rat tissues.

Rabbit antiserum against rat liver-soluble cytochrome b9 was produced. The antiserum was specific to cytochrome b9 and did not react with hemoglobin, cytochrome c, cytochrome H-450, serum, hemolysate, and tissue extracts from lung, brain, testis, and skeletal muscle. From Ouchterlony double immunodiffusion and tandem crossed immunoelectrophoresis, the antigen was found in liver, kidney, and heart tissue extracts of rat with complete identity. From crossed immunoelectrophoresis, the antiserum distinguished monomeric and dimeric forms of the antigen. Rocket immunoelectrophoresis for quantitating the antigen was developed with a minimum sensitivity of 5 ng. By using rocket immunoelectrophoresis, the concentration of the antigen in crude tissue extracts from liver, heart, and kidney was determined. The antigen was not detectable in crude extracts from lung, brain, testis, and skeletal muscle by immunological methods. Therefore, the immunological assay method was very useful for identifying and quantitating cytochrome b9 in crude extracts with high sensitivity.

Animals↗
PubMed1979

Isolation and partial characterization of a new soluble b-type cytochrome (b9) from rat liver.

A new soluble cytochrome, designated as cytochrome b9, was purified to apparent homogeneity from rat liver. The absorption maximum of the oxidized (the native form) cytochrome b9 at room temperature was 413 nm. The dithionite-reduced cytochrome b9 had absorption maxima at 556, 527, and 423 nm. The prosthetic group of cytochrome b9 was identified as protoheme IX. From gel filtration experiments, the molecular weight of cytochrome b9 was estimated to be 125,000. Polyacrylamide gel electrophoresis experiments in the presence of sodium dodecyl sulfate showed that the molecular weight of its subunit was 61,000. The native form of cytochrome b9 was thus a dimer. The amount of heme/mol of dimer was 3.3 mol. Cytochrome b9 was autoxidizable and did not bind CO, 2.2 mM cyanide, or 2.2 mM azide. On the basis of its molecular weight of 125,000, the millimolar extinction coefficients of dimeric cytochrome b9 at 280 and 413 nm were 384 and 380, respectively. The absorbance at 280 nm/mg cytochrome b9 was 3.1. Cytochromes b9 and H-450 (I.-C. Kim and W.C. Deal (1976) Biochemistry 15, 4925-4930) are the only b-type, soluble cytochromes which have been isolated from mammalian liver; they are not found in tissues of heart, lung, kidney, and brain. The biological function of cytochrome b9 was not determined.

Animals↗
PubMed1976

Isolation and properties of a new, soluble, hemoprotein (H-450) from pig liver.

A new soluble hemoprotein, designated as H-450, has been purified from pig liver. The absolute absorption spectrum of H-450 shows maxima at 550 and 428 nm. The dithionite-reduced H-450 has absorption peaks at 572, 540, and 450 nm; the unique Soret band at 450 nm is the basis for our tentative designation of this new hemoprotein as H-450 (hemoprotein 450). The spectrum of dithionite-reduced H-450 at 77 K gives two alpha peaks (571 and 566 nm), three beta peaks (546, 537, and 529 nm), and a Soret band at 449 nm. The prosthetic group of H-450 has been identified as protoheme IX. Gel electrophoresis experiments show that H-450 is composed of two nonidentical subunits, alpha and beta (mol wts = 61 000 and 45 000). H-450 contains 1 mol of heme/alphabeta dimer of 106 000 molecular weight. Preliminary sedimentation equilibrium experiments suggest a minimum molecular weight of 218 000 for the native protein. This corresponds to a tetramer, alpha2beta2 containing two heme groups. H-450 is not reduced by reduced nicotinamide adenine dinucleotide (NADH), NADH phosphate, ascorbate, or ferrocyanide. Neither reduced nor oxidized H-450 binds CO, 1 mM cyahide, or 1 mM azide. Dithionite-reduced H-450 is autoxidizable. The molar extinction coefficient of native H-450 is 261 X 103 at 280 nm and 263 X 103 at 428 nm. The purification procedure involves homogenization, high-speed centrifugation, ammonium sulfate fractionation, diethylaminoethylcellulose chromatography, density gradient centrifugation, a calcium phosphate gel step, and a second density gradient centrifugation. The procedure yeilds approximately 2 mg of purified protein from 750 g of pig liver.

Animals↗