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Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b5
95
Biochimica et Biophysica Acta, 1172 (1993) 95-100 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 92466
Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b 5 Sara J. Giordano and Alan W. Steggles Department of Biochemistry and Molecular Pathology, Northeastern Ohio UniversitiesCollege of Medicine, Rootstown, OH (USA) (Received 23 July 1992)
Key words: Cytochrome bs; Genetics; Expression; Distribution
Total RNA was extracted from a variety of rabbit tissues and reverse transcribed for use in the polymerase chain reaction technique. Using primers designed to amplify the membrane-bound liver cytochrome b 5 cDNA, products of two sizes were observed. Both hybridized strongly to a radiolabelled liver cytochrome b 5 probe. Sequencing confirmed that the two types of cDNA product encoded the membrane-bound and the soluble forms of b 5. Messenger RNA corresponding to the soluble cytochrome was detected in the lung, gallbladder and the adrenal gland, as well as in reticulocytes and bone marrow. This was an unexpected finding since the protein has been isolated only from erythrocytes. In contrast, membrane-bound cytochrome b 5 mRNA was detected in all tissues tested, suggesting that the corresponding protein is ubiquitous in tissue distribution.
Introduction
Cytochrome b 5 is a small hemeprotein which participates in electron transfer in a variety of biochemical reactions, including the NADH-dependent reduction of methemoglobin to hemoglobin in erythrocytes [1], as well as fatty acid desaturation [2,3l, fatty acid elongation [4] and P-450 reduction [5] in liver and other tissues. The protein has been isolated from the liver and erythrocytes of several species [6-16] in two distinct forms. One form of cytochrome b 5 (b~) is a soluble protein composed of 98 amino acids (counting the initial methionine as number one). The other form is an integral protein of the endoplasmic reticulum and consists of 134 amino acids which comprise two functional domains. The hydrophobic, carboxy-terminal end of the protein serves as a membrane anchor, while the hydrophilic, amino-terminal portion forms the hemebinding, catalytic domain. Protein sequencing has confirmed that the soluble, erythrocyte form and the membrane-bound, liver form are identical in amino acid sequence for residues 1 to
Correspondence to: A.W. Steggles, Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, P.O. Box 95, Rootstown, OH 44272, USA. The accession numbers to the EMBL/Genbank Data Libraries for the rabbit membrane bound and soluble forms of b 5 are M24844 and Z14091, respectively.
97 in all species studied. This finding suggested that the smaller cytochrome b 5 may arise as a result of proteolytic cleavage of the membrane-bound form. However, amino acid 98 of erythrocyte cytochrome b 5 is different from that of the liver form in all but one species. This suggests that the two cytochromes are derived from two nearly identical genes, or from a common precursor m R N A through alternative splicing mechanisms. The nucleotide sequences of liver b 5 mRNAs have been determined for the chicken, cow, rabbit and human [17-21]. Additionally, erythrocyte b5 m R N A was found to be identical in sequence to liver b 5 m R N A in the chicken [17]. In contrast, human liver and erythrocyte b5 mRNAs were found to be highly homologous, but different, due to alternative splicing [22]. The apparent incongruity of the human and chicken findings reflects a major difference in erythrocytes from the two species: avian red blood cells are nucleated while mammalian cells are devoid of a nucleus, endoplasmic reticulum and other organelles. Cytochrome b5 has been detected in a variety of tissues. Spectrophotometric analysis of microsomes indicated that membrane-bound b 5 was present in liver, kidney, spleen, fat, gonads, adrenals, stomach, thymus, lung, brain, pancreas and embryonic muscle, but was absent in adult muscle [23]. The spectral properties of a crude homogenate of pig kidney suggested that soluble b 5 was also present in this tissue [24]. However, the molecular weight of the partially purified protein was
96 consistent with that of membrane-bound b 5. A determination of redox potentials in liver and brain microsomes provided evidence for the presence of two bss in the brain, both of which were different from the liver cytochrome [25]. Northern blot analysis has demonstrated the presence of membrane-bound b.~ mRNA in human liver [21] and in chicken erythrocytes and liver [17] but not in other tissues. Soluble b~s mRNA is rare in reticulocytes [22] and has eluded detection by Northern analysis. Because the polymerase chain reaction (PCR) is a highly sensitive technique [26,27] and has been used successfully to study rare mRNAs such as dystrophin [28], we chose to employ this method for determining the tissue localization of b_s mRNAs in the rabbit. We were particularly interested in determining (a) if soluble b~ was present in tissues other than erythrocytes, such as kidney, (b) if other homologous forms of b5 existed in the brain and (c) the presence or absence of b5 mRNA in adult muscle. Finally, we wanted to learn if rabbit erythrocyte b_s was the product of post-transcriptional or of post-translational processing, by sequencing reticulocyte mRNA.
Tissue collection
Materials and Methods
Leukocytes and reticulocytes were isolated from 2 ml of heparinized blood obtained from an anesthetized, New Zealand white rabbit by cardiac puncture. Following centrifugation of the whole blood at 1000 × g for 10 min, the leukocyte-rich buffy coat was transferred to a 1.5 ml Eppendorf tube containing 1.0 ml of an erythrocyte-lysing solution, 0.9% ammonium chloride. Approx. 0.5 ml of the reticulocyte-rich red cell layer below the buffy coat was transferred to another 1.5 ml Eppendorf tube containing 1.0 ml of PBS (138 mM NaC1, 2.7 mM KC1, 10 mM N a : H P O 4, 1.8 mM KH2PO 4, pH 7.4). The tubes were centrifuged at 6000 × g for 1 min and the resultant supernatants were discarded. The reticulocyte and leukocyte suspensions were washed three additional times with PBS. To eliminate leukocytes from the reticulocyte suspension, the uppermost 100 /zl of red cells were removed and discarded from that tube during each wash. Purity of the erythrocyte population was determined by microscopic examination of a Wright's stained smear of cells. Other tissue samples, each weighing approx. 0.05 g, were collected from a euthanized, adult rabbit. The specimens were used immediately or stored at minus 70°C for up to 6 months prior to testing.
Chemicals"
R N A extraction
Chemicals of the highest quality available were purchased from the following sources: guanidinium thiocyanate was from Fluka (Ronkonkoma, NY); sarcosyl, sodium citrate, sodium chloride, potassium chloride, monobasic potassium phosphate, dibasic sodium phosphate, sodium acetate, magnesium chloride, ammonium acetate, Trizma base, ethanol, isoproopanol, phenol, chloroform, EDTA, boric acid and 2-mercaptoethanol were from Fisher Scientific (Fair Lawn N J); DECAprime oligolabelling kit was from Ambion (Austin, TX); Amplitaq Polymerase, PCR buffer, and deoxynucleotide triphosphates (dNTPs) were from Perkins-Cetus (Norwalk, CT); Sequenase and sequencing reagents were from United States Biochemical (Cleveland, OH); random hexamers were from Boehringer-Mannheim (Indianapolis, IN); Maloney murine leukemia virus reverse transcriptase (RT), and RT buffer were from Bethesda Research Laboratories (Gaithersburg, MD); RNAsin was from Promega (Madison, WI); [ce-32p]dATP, [a-32p]dCTP and Genescreen Plus nylon membrane were from New England Nuclear (Boston, MA); Agarose and Nu-Sieve G T G were from FMC BioProducts (Rockland, ME); nonpurified oligonucleotides were custom synthesized by National Biosciences (Plymouth, MN). Reagents were used according to the manufacturer's instructions except as noted. Procedures and techniques employed were those described by Sambrook et al. [29], unless otherwise indicated.
A modification of the acid guanidinium thiocyanate-phenol method described by Chomczynski and Sacchi [30] was used for RNA extraction. Frozen tissue was added to 500/zl guanidinium thiocyanate solution (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 0.1 M 2-rnercaptoethanol), and immediately homogenized using a polytron. Then 0.1 ml of 2 M sodium acetate (pH 4.0), 0.5 ml of water saturated phenol and 0.1 ml of chloroform were added to the suspension. After vortexing for 10 s and incubating on ice for 15 min, the samples were centrifuged at 12000 × g for 10 rain. The aqueous phase was transferred to a new tube and the phenol-chloroform extraction was repeated twice. RNA was precipitated with isopropanol, redissolved in 0.3 ml guanidinium thiocyanate and then reprecipitated in isopropanol containing 0.2 M sodium acetate (pH 4.0). The resulting pellet was washed twice with 70% ethanol and dried under vacuum. Rer, erse transcription
Total RNA was resuspended in a final volume of 32 /zl containing 10/zl of 5 × RT buffer and 500 pmol of random hexamers. The mixture was incubated at 100°C for 2 min and stored on ice. The volume was adjusted to 50 /zl such that the final mixture contained 1000 units reverse transcriptase, 200 /~M dNTPs, 10 mM D T T and 20 U of RNAsin. The mixture was incubated at room temperature for 10 rain then at 42°C for 50
97 min. The enzyme was inactivated by incubation at 68°C for 10 rain.
uration at 94°C for 1.5 min, annealing at 50°C (using primers 2 and 3) or 60°C (using all other primer combinations) for 2.5 min and extension at 72°C for 2 min. A negative control containing all of the reagents except DNA template was run with each experiment.
Oligonucleotides The sequences of the primers and the amino acids (aa) of the liver or reticulocyte form to which they correspond are as follows: primer 1, G A G A T G G C T G C G C A G T C A G A C , aa - 1 to + 6; primer 2, G G A G G A A G T C C T G A G G G A A C , aa 47-53; primer 3, CTTG A A A A C A G A C C T T , genomic sequence of a putative reticulocyte exon; primer 4, T C A C C C A G T T G G T C CACCA, aa 114-109; primer 5, G G C T G C T G G G C A G G G G C T C A , 3' non-translated to aa 134.
Analysis of PCR products One-third of each reaction was analyzed on a 1.6% agarose gel which was subsequently transferred to a nylon membrane by Southern blotting [29] and hybridized to an [a-32P]dCTP-oligolabelled rabbit liver cytochrome b 5 probe [19] at 65°C using Church's hybridization solution [30]. Membranes were exposed to X-ray film for 12-24 h. Some PCR products were isolated on low-melt agarose for use in reamplification reactions.
PCR amplification Amplification of 1.0 /xl of the reverse transcribed total RNA was carried out in a final volume of 50/~1 containing 200 /xM dNTPs, 0.1 /~M each of forward and reverse primers, 0.25 U / m l Taq Polymerase and buffer at a final concentration of 50 mM KC1, 10 mM Tris (pH 8.3), 1.5 mM MgC12 and 0.1% (w/v) gelatin. The mixture was overlaid with mineral oil. An MJ Research (Watertown, MA) thermal cycler was used to carry out 35 cycles of amplification consisting of denat-
A
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5
Asymmetric PCR Approx. 4 ng (2 /zl) of cDNA in low melt agarose was neutralized with MgC12 and used for asymmetric PCR [32] reamplification. Conditions for PCR were those described above, except that the ratio of primers was 1:100 with the predominant primer at a final concentration of 0.1 ~zM.
6
7
8
9
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I
10 11
I
12
13
14
15
16
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18 19
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B
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D
Fig. 1. Agarose gel electrophoresis (a and c) and Southern blots (b and d) of cytochrome b 5 PCR products. Lane assignment is by tissue source: 1, reticulocytes; 2, leukocytes; 3, liver; 4, heart; 5, spleen; 6, pancreas; 7, ovary; 8, adrenal gland; 9, uterus; 10, femoral muscle; 11, soleus muscle; 12, bone; 13, bone marrow; 14, brain; 15, lung; 16, fat; 17, kidney; 18, gallbladder; 19, negative control. Primers were designed to amplify both the membrane-bound and the soluble forms of cytochrome b 5 mRNA (a and b), or the soluble form (c and d) only. In (a) and (b), a 203 bp product, corresponding to membrane-bound cytochrome b_s cDNA, appears in all of the tissues specimens. A slightly larger, 231 bp product, representing soluble cytochrome bs, is visualized in reticulocytes, adrenal gland, bone marrow, lung and gallbladder. The high molecular weight product seen in lanes 1, 8, 13, 15 and 18 in (a) and (b) is a PCR artifact which yields the lower molecular weight products upon reamplification. In (c) and (d), the presence of soluble b_s mRNA is confirmed in reticulocytes, adrenal gland, bone marrow, lung and gallbladder.
98
Sequencing The products of asymmetric PCR were precipitated with one-forth volume of 10 M ammonium acetate and an equal volume of isopropanol. The resultant pellets were washed twice in 70% ethanol and dried under vacuum. Direct sequencing was carried out by the dideoxy chain termination method of Sanger [33] using the single-stranded DNA from asymmetric PCR as template. Results
PCR determination of cytochrome b5 mRNA distribution A total of 18 tissues were initially selected for analysis. These included erythrocytes, leukocytes, liver, heart, spleen, pancreas, ovary, adrenal gland, uterus, fasttwitch muscle, slow-twitch muscle, bone, bone marrow, brain, lung, fat, kidney and gallbladder. Total RNA was isolated from each tissue sample, reverse-transcribed and used directly in PCR reactions. The availability of rabbit b5 genomic sequence (unpublished) and mRNA sequences for human liver and reticulocyte bss [20,22] allowed us to speculate that the mRNAs for soluble and membrane-bound rabbit bss would be homologous except for the inclusion of an additional, internal exon in the soluble b 5 mRNA. Based on the published sequence of rabbit liver cytochrome b5 m R N A [19], primer 1 (forward) and primer 5 (reverse) were designed to amplify the coding region of either mRNA. Following 35 cycles of PCR amplification the products were analyzed on agarose gels. Many of the samples appeared as smears rather than as discrete bands. This was not a totally unexpected finding, since b 5 is a member of a bs-like superfamily of proteins which includes sulfite oxidase, flavocytochrome b 2, nitrate reductase, and outer mitochondrial b5 [34]. All of these cytochromes exhibit approx. 30 to 80 percent homology to microsomal b5 in their catalytic domains and may, therefore, compete nonspecifically with b5 for primer binding. In order to increase target specificity, primer 2 (forward) and primer 4 (reverse) were constructed to amplify a smaller segment of the coding region. As seen in Fig. la, use of these primers for PCR amplification resulted in product formation from all of the tissue cDNAs. In addition, a product of larger size was apparent in five of the samples. The gel was transferred by Southern blotting and hybridized under stringent conditions to a radiolabelled rabbit liver b 5 cDNA probe. The results, as shown in Fig. lb, indicate that the products were specific for cytochrome b~. The larger-sized PCR product was believed to correspond to the putative soluble bs. However, the possibility existed that it represented a new cytochrome b5 sequence. To clarify the results, primer 3 was designed. Analysis of rabbit genomic sequence (unpublished)
primer i GAG ATG ~CT GC~ CAG TCA GAC AAA GAC GTG AAG TAC TAC ACC CTA met ala ala gln ser asp lys asp val lys tyr tyr thr leu
(14)
GAA GAG ATT AAG AAG CAC AAC CAC AGC AAA AGC ACC TGG CTG ATC glu glu ile lys lys his asn his s e r l y s s e r thr trp leu ile
(29)
CTG CAC CAC AAG GTG TAC GAT leu his his lys val try asp pr~m~ 2 CCT GGA GGG GAG GAA GTC CTG pro gly gly glu glu val leu
CTG ACC AAA TTT CTG GAG GAG CAC leu thr lys phe leu glu glu his
(44)
AGG GAA CAA GCT GGG GGC GAT GCC arg glu gln ala gly gly asp ala
(59)
ACT GAA AAC TTT GAG GAC GTC GGG CAC TCG ACA GAT GCC AGA GAG thr glu asn phe glu asp val gly his ser thr asp ala arg glu
(74)
CTG TCC AAG ACC TTC ATC ATC GGG GAG CTG CAC CCG GAT GAC AGA leu ser lys thr phe ile ile gly glu leu his pro asp asp arg
(89)
TCA AAA TTG AGC AAG CCT ATG ser lys leu s e r lys pro met
(96)
GAA ACT CTT glu thr leu pr~m~r ~ AAC TGG GTG asn trp val
IGAA C~T T A A A G G C T G T G T T T C ~ A G I {glu pro stop [ 9 8 } I
ATC ACC ACC GTC GAT TCC AAT TCC AGC TGG TGG ACC ile thr thr val asp ser asn ser ser trp trp thr (iii)
ATC CCC GCC ATC TCC GCC CTG ATC GTG GCA CTG ATG ile pro ala ile ser ala leu ile val ala leu met (126) pr~me~ 5 TAT CGC CTC TAC ATC CCC GAC GAC T~A GCCCCT~CCCAGCACCC (134) tyr arg leu try met ala asp asp stop
Fig. 2. Nucleotide sequence and deduced amino acid sequences for the m e m b r a n e - b o u n d b 5 c D N A from brain and soluble b5 c D N A from reticulocytes. Regions of homology to the oligonucleotide primers are underlined. The two sequences are identical except for the inclusion of an additional 24 bp exon (in bold type and brackets) in the reticulocyte sequence.
suggested a potential exon for soluble b5 mRNA. A sequence immediately 3' to the proposed exon was selected for use as primer 3. Fig. lc shows the results of the PCR amplification using primer 2 and primer 3. Cytochrome b5 specificity was confirmed by hybridization of a Southern blot. In addition, DNA sequencing established that all five products in Fig. lc corresponded to the soluble form of b 5.
Determination of the complete cytochrome b5 sequences The PCR products contained about 200 of the 400 or more possible bp of coding information. In order to obtain maximal sequence information, brain and bone marrow specimens were subjected to PCR amplification using primers 1 and 4 to generate the 5' coding region, or primers 2 and 5 to generate the 3' coding region. The products were isolated on low melt agarose and used as seed for asymmetric PCR reamplification reactions. The resultant single-stranded cDNAs were directly sequenced. The composite sequences from the brain and bone marrow cDNAs are shown in Fig. 2. Discussion
The results presented here demonstrate that rabbit soluble cytochrome b 5 is encoded by a mRNA that is homologous to liver b.~ mRNA, but which differs in length by 24 bp due to the inclusion of an internal exon. This 24 bp sequence is the same as that described by Takematsu et al. [35] although they had misaligned their sequence by 3 bp, because they did
99 not have the genomic sequence to specifically localize the exon. Although soluble b 5 m R N A is longer than membrane-bound b 5 mRNA, it encodes a smaller protein as a result of a termination codon in the additional exon. Our data suggest that both membrane-bound and soluble b 5 mRNAs are derived from a common precursor m R N A by alternative splicing mechanisms in a manner analagous to that shown for human b 5 [22]. The sequence of the extra 24 bp found in the soluble b5 m R N A differed from the sequence previously obtained from rabbit genomic DNA. The PCR primer 3, derived from this genomic sequence, functioned in a specific manner even though it contained five misalligned nucleotides. It has been shown previously that only the first three 5' nucleotides are absolutely critical for primer specificity, and that the primer will bind as long as there is sufficient homology in the rest of the primer sequence [36]. Based on our results, we conclude that the tissue distribution of membrane-bound cytochrome b 5 mRNA is probably ubiquitous (Fig. la,b). Because adult, skeletal muscle was previously shown to lack bs, we initially selected two muscles, the soleus and the femoral, for testing. As shown in Fig. la,b (lanes 10, 11), both samples displayed b5 mRNA. We confirmed our findings by harvesting and testing RNA isolated from six additional, different muscle specimens. All of the samples exhibited the membrane-bound b5 m R N A (results not shown). Although it could be argued that the mRNA that we detected was from leukocytes in the capillaries of the muscles, the absence of soluble b5 PCR product negates the possibility since reticulocytes are present in equal or greater number than leukocytes in whole blood. Our conclusions regarding muscle b5 are not necessarily in opposition to previous findings [23], but rather, reflect the high degree of sensitivity that can be attained with PCR techniques. We did not expect to find membrane-bound b 5 m R N A in reticulocytes. The presence of both forms of b 5 m R N A in these cells suggests that both post-transcriptional and post-translational processing are involved in the formation of soluble bs. An alternative explanation for the finding of membrane-bound b5 mRNA in reticulocytes is that the erythrocyte samples used in this study contained leukocytes. Although stained smears of the erythrocyte suspensions provided no evidence for contamination by white ceils, the sensitivity of PCR is such that the presence of one leukocyte in the sample would result in a highly visible product corresponding to membrane-bound b 5. Prior to this study, the presence of soluble cytochrome b 5 had been confirmed in erythrocytes, and suggested in kidney. The present findings (Fig. lc,d) reaffirm the presence of soluble b5 mRNA in reticulocytes and in bone marrow, the tissue in which reticulocytes are produced. The additional detection of solu-
ble b 5 m R N A in the lung, gallbladder and adrenal gland was an unexpected finding, since the soluble form of the protein was thought to function exclusively in methemoglobin reduction. These results suggest that the protein may also participate in cytoplasmic redox reactions which have not yet been elucidated. Although it could be argued that the finding of soluble b5 in the lung, gallbladder and adrenal gland is due to contamination of the tissues with reticulocytes, the absence of soluble b~ in the highly-vascularized spleen, liver and kidney refutes the argument. Considering the inherent sensitivity of PCR, we do not believe that soluble b~ is present in kidney, but we cannot exclude the possibility that tissue distribution differs in the rabbit and the pig. The use of primers 2 and 3 (Fig. lc,d) for the specific amplification of the reticulocyte b~ sequence gave results consistant with those shown in Fig. la,b. The increased levels of primer dimer product visible in Fig. ld is probably due to primer 3 not being a perfect match with the actual reticulocyte specific b~ sequence. DNA sequencing confirmed that the major signals in Fig. ld corresponded to soluble cytochrome b5 cDNA. We were unable to confirm the presence of a new, homologous b~ in brain tissue, even though the methods employed in this study allowed us to detect both the soluble and membrane-bound cytochrome b~ mRNAs. We chose to directly sequence the PCR product in order to detect any differences in m R N A sequence which might correspond to a new b 5. The nucleotide sequence of brain b 5 was identical to that of liver b~. It is unlikely that our detection of this form was due to contamination by blood pooled in capillaries because no reticulocyte form was observed. Our results suggest that the brain contains only one form of membranebound cytochrome b~ and that it is identical to that found in liver. However, the apparent conflict between our findings and the potentiometric evidence for additional forms of b~ in brain microsomes [25], may reflect changes in the protein due to post-translational processing of membrane-bound cytochrome bs.
Acknowledgement This work was supported by a grant from the National American Heart Association and its Ohio affiliate.
References 1 Hultquist, D.E. and Passon, P.G. (1971) Nature New. Biol. 229, 252-254. 2 Jones, P.D., Holloway,P.W., Peluffo, R.O. and Wakil, A.J. (1969) J. Biol. Chem. 244, 744-754. 30shino, N. and Omura, T. (1973) Arch. Biochem. Biophys. 157, 395-404. 4 Keyes, S.R. and Cinti, D.L. (1980) J. Biol. Chem. 255, 1135711364.
100 5 White, R.E. and Coon, M.J. (1980) Annu. Rev. Biochem. 49, 315-356. 6 0 z o l s , J. and Gerard, C. (1977) Proc. Nat~. Acad. Sci. USA 74, 3725-3729. 7 Kimura, S., Abe, K. and Sugita, Y. (1984) FEBS Lett. 169, 143-146. 8 Ozols, J. and Heinemann, F.S. (1982) Biochim. Biophys. Acta 704, 163-173. t,} Ozols, J. and Gerard, C. (1977) J. Biol. Chem. 252, 8549-8553. 10 Fleming, P.J., Dailey, H.A., Corcoran, D. and Strittmatter, P. (1978) 253, 5369-5372. 11 Ozols, J. (1972) J. Biol. Chem. 247, 2242-2254. 12 Ozols, J. (1970) J. Biol. Chem. 345, 4863-4874. 13 Nobrega, F.G. and Ozols, J. (1971)J. Biol. Chem. 246, 1706-1717. 14 Passon, P.G., Reed, D.W. and Hultquist, D.E. (1972) Biochim. Biophys. Acta 275, 51-61. 15 Slaughter, S.R., Williams, C.H. and Hultquist, D.E. (1982) Biochim. Biophys. Acta 705, 228-237. 16 Schafer, D.A. and Hultquist, D.E. (1983) Biochem. Biophys. Res. Commun. 115, 807-813. 17 Zhang, H. and Somerville, C. (1990) Arch. Biochem. Biophys. 280, 412-415. 18 Cristiano. R.J. and Steggles, A.W. (1989) Nucleic Acids Res. 17, 799. 19 Dariush, N., Fisher, C.W. and Steggles, A.W. (1988) Prot. Seq. Data Anal. 1~ 351-353. 2(t Yoo, M. and Steggles, A.W. (1988) Biochem. Biophys. Res. Commun. 156, 576-580. 21 Miyata, M., Nagata, K., Yamazoe, Y. and Kato, R. (1989) Pharm. Res. 21,513-519.
22 Giordano, S.J. and Steggles, A.W.(1991) Biochem. Biophys. Res. Commun. 178, 38-44. 23 Garfinkel, D. (1963) Comp. Biochem. Physiol. 8, 367-379. 24 Mangum, J.H., Klingler, M.D. and North, J.A. (1970) Biochem. Biophys. Res. Commun. 40, 1520-1525. 25 Yoshida, S., Yubisui, T. and Takeshita, M. (1984) Arch. Biochem. Biophys. 232, 296-304. 26 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Science 239, 487-491. 27 Saiki, R.K., Schaff, S., Faloona, F., Mullis, K.B., Horn, G.T., Ehrlich, H.A. and Arnheim, N. (1985) Science 230, 1350-1354. 28 Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A. (1988) Nature 333, 858-860. 29 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 30 Church, G.M. and Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991 1995. 31 Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156 159. 32 Gyllensten, U. and Erlich, H.A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652 7656. 33 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 34 Mathews, F.S, (1985) Prog. Biophys. Mol. Biol. 45~ 1-56. 35 Takamatsu, H., Kozutsumi, Y., Suzuki, A. and Kawasaki, T. (1992) Biochem. Biophys. Res. Commun. 185, 845-851. 36 Sommer, R. and Tautz, D. (1989) Nucleic Acids Res. 17, 6749.
Biochimica et Biophysica Acta, 1172 (1993) 95-100 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 92466
Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b 5 Sara J. Giordano and Alan W. Steggles Department of Biochemistry and Molecular Pathology, Northeastern Ohio UniversitiesCollege of Medicine, Rootstown, OH (USA) (Received 23 July 1992)
Key words: Cytochrome bs; Genetics; Expression; Distribution
Total RNA was extracted from a variety of rabbit tissues and reverse transcribed for use in the polymerase chain reaction technique. Using primers designed to amplify the membrane-bound liver cytochrome b 5 cDNA, products of two sizes were observed. Both hybridized strongly to a radiolabelled liver cytochrome b 5 probe. Sequencing confirmed that the two types of cDNA product encoded the membrane-bound and the soluble forms of b 5. Messenger RNA corresponding to the soluble cytochrome was detected in the lung, gallbladder and the adrenal gland, as well as in reticulocytes and bone marrow. This was an unexpected finding since the protein has been isolated only from erythrocytes. In contrast, membrane-bound cytochrome b 5 mRNA was detected in all tissues tested, suggesting that the corresponding protein is ubiquitous in tissue distribution.
Introduction
Cytochrome b 5 is a small hemeprotein which participates in electron transfer in a variety of biochemical reactions, including the NADH-dependent reduction of methemoglobin to hemoglobin in erythrocytes [1], as well as fatty acid desaturation [2,3l, fatty acid elongation [4] and P-450 reduction [5] in liver and other tissues. The protein has been isolated from the liver and erythrocytes of several species [6-16] in two distinct forms. One form of cytochrome b 5 (b~) is a soluble protein composed of 98 amino acids (counting the initial methionine as number one). The other form is an integral protein of the endoplasmic reticulum and consists of 134 amino acids which comprise two functional domains. The hydrophobic, carboxy-terminal end of the protein serves as a membrane anchor, while the hydrophilic, amino-terminal portion forms the hemebinding, catalytic domain. Protein sequencing has confirmed that the soluble, erythrocyte form and the membrane-bound, liver form are identical in amino acid sequence for residues 1 to
Correspondence to: A.W. Steggles, Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, P.O. Box 95, Rootstown, OH 44272, USA. The accession numbers to the EMBL/Genbank Data Libraries for the rabbit membrane bound and soluble forms of b 5 are M24844 and Z14091, respectively.
97 in all species studied. This finding suggested that the smaller cytochrome b 5 may arise as a result of proteolytic cleavage of the membrane-bound form. However, amino acid 98 of erythrocyte cytochrome b 5 is different from that of the liver form in all but one species. This suggests that the two cytochromes are derived from two nearly identical genes, or from a common precursor m R N A through alternative splicing mechanisms. The nucleotide sequences of liver b 5 mRNAs have been determined for the chicken, cow, rabbit and human [17-21]. Additionally, erythrocyte b5 m R N A was found to be identical in sequence to liver b 5 m R N A in the chicken [17]. In contrast, human liver and erythrocyte b5 mRNAs were found to be highly homologous, but different, due to alternative splicing [22]. The apparent incongruity of the human and chicken findings reflects a major difference in erythrocytes from the two species: avian red blood cells are nucleated while mammalian cells are devoid of a nucleus, endoplasmic reticulum and other organelles. Cytochrome b5 has been detected in a variety of tissues. Spectrophotometric analysis of microsomes indicated that membrane-bound b 5 was present in liver, kidney, spleen, fat, gonads, adrenals, stomach, thymus, lung, brain, pancreas and embryonic muscle, but was absent in adult muscle [23]. The spectral properties of a crude homogenate of pig kidney suggested that soluble b 5 was also present in this tissue [24]. However, the molecular weight of the partially purified protein was
96 consistent with that of membrane-bound b 5. A determination of redox potentials in liver and brain microsomes provided evidence for the presence of two bss in the brain, both of which were different from the liver cytochrome [25]. Northern blot analysis has demonstrated the presence of membrane-bound b.~ mRNA in human liver [21] and in chicken erythrocytes and liver [17] but not in other tissues. Soluble b~s mRNA is rare in reticulocytes [22] and has eluded detection by Northern analysis. Because the polymerase chain reaction (PCR) is a highly sensitive technique [26,27] and has been used successfully to study rare mRNAs such as dystrophin [28], we chose to employ this method for determining the tissue localization of b_s mRNAs in the rabbit. We were particularly interested in determining (a) if soluble b~ was present in tissues other than erythrocytes, such as kidney, (b) if other homologous forms of b5 existed in the brain and (c) the presence or absence of b5 mRNA in adult muscle. Finally, we wanted to learn if rabbit erythrocyte b_s was the product of post-transcriptional or of post-translational processing, by sequencing reticulocyte mRNA.
Tissue collection
Materials and Methods
Leukocytes and reticulocytes were isolated from 2 ml of heparinized blood obtained from an anesthetized, New Zealand white rabbit by cardiac puncture. Following centrifugation of the whole blood at 1000 × g for 10 min, the leukocyte-rich buffy coat was transferred to a 1.5 ml Eppendorf tube containing 1.0 ml of an erythrocyte-lysing solution, 0.9% ammonium chloride. Approx. 0.5 ml of the reticulocyte-rich red cell layer below the buffy coat was transferred to another 1.5 ml Eppendorf tube containing 1.0 ml of PBS (138 mM NaC1, 2.7 mM KC1, 10 mM N a : H P O 4, 1.8 mM KH2PO 4, pH 7.4). The tubes were centrifuged at 6000 × g for 1 min and the resultant supernatants were discarded. The reticulocyte and leukocyte suspensions were washed three additional times with PBS. To eliminate leukocytes from the reticulocyte suspension, the uppermost 100 /zl of red cells were removed and discarded from that tube during each wash. Purity of the erythrocyte population was determined by microscopic examination of a Wright's stained smear of cells. Other tissue samples, each weighing approx. 0.05 g, were collected from a euthanized, adult rabbit. The specimens were used immediately or stored at minus 70°C for up to 6 months prior to testing.
Chemicals"
R N A extraction
Chemicals of the highest quality available were purchased from the following sources: guanidinium thiocyanate was from Fluka (Ronkonkoma, NY); sarcosyl, sodium citrate, sodium chloride, potassium chloride, monobasic potassium phosphate, dibasic sodium phosphate, sodium acetate, magnesium chloride, ammonium acetate, Trizma base, ethanol, isoproopanol, phenol, chloroform, EDTA, boric acid and 2-mercaptoethanol were from Fisher Scientific (Fair Lawn N J); DECAprime oligolabelling kit was from Ambion (Austin, TX); Amplitaq Polymerase, PCR buffer, and deoxynucleotide triphosphates (dNTPs) were from Perkins-Cetus (Norwalk, CT); Sequenase and sequencing reagents were from United States Biochemical (Cleveland, OH); random hexamers were from Boehringer-Mannheim (Indianapolis, IN); Maloney murine leukemia virus reverse transcriptase (RT), and RT buffer were from Bethesda Research Laboratories (Gaithersburg, MD); RNAsin was from Promega (Madison, WI); [ce-32p]dATP, [a-32p]dCTP and Genescreen Plus nylon membrane were from New England Nuclear (Boston, MA); Agarose and Nu-Sieve G T G were from FMC BioProducts (Rockland, ME); nonpurified oligonucleotides were custom synthesized by National Biosciences (Plymouth, MN). Reagents were used according to the manufacturer's instructions except as noted. Procedures and techniques employed were those described by Sambrook et al. [29], unless otherwise indicated.
A modification of the acid guanidinium thiocyanate-phenol method described by Chomczynski and Sacchi [30] was used for RNA extraction. Frozen tissue was added to 500/zl guanidinium thiocyanate solution (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 0.1 M 2-rnercaptoethanol), and immediately homogenized using a polytron. Then 0.1 ml of 2 M sodium acetate (pH 4.0), 0.5 ml of water saturated phenol and 0.1 ml of chloroform were added to the suspension. After vortexing for 10 s and incubating on ice for 15 min, the samples were centrifuged at 12000 × g for 10 rain. The aqueous phase was transferred to a new tube and the phenol-chloroform extraction was repeated twice. RNA was precipitated with isopropanol, redissolved in 0.3 ml guanidinium thiocyanate and then reprecipitated in isopropanol containing 0.2 M sodium acetate (pH 4.0). The resulting pellet was washed twice with 70% ethanol and dried under vacuum. Rer, erse transcription
Total RNA was resuspended in a final volume of 32 /zl containing 10/zl of 5 × RT buffer and 500 pmol of random hexamers. The mixture was incubated at 100°C for 2 min and stored on ice. The volume was adjusted to 50 /zl such that the final mixture contained 1000 units reverse transcriptase, 200 /~M dNTPs, 10 mM D T T and 20 U of RNAsin. The mixture was incubated at room temperature for 10 rain then at 42°C for 50
97 min. The enzyme was inactivated by incubation at 68°C for 10 rain.
uration at 94°C for 1.5 min, annealing at 50°C (using primers 2 and 3) or 60°C (using all other primer combinations) for 2.5 min and extension at 72°C for 2 min. A negative control containing all of the reagents except DNA template was run with each experiment.
Oligonucleotides The sequences of the primers and the amino acids (aa) of the liver or reticulocyte form to which they correspond are as follows: primer 1, G A G A T G G C T G C G C A G T C A G A C , aa - 1 to + 6; primer 2, G G A G G A A G T C C T G A G G G A A C , aa 47-53; primer 3, CTTG A A A A C A G A C C T T , genomic sequence of a putative reticulocyte exon; primer 4, T C A C C C A G T T G G T C CACCA, aa 114-109; primer 5, G G C T G C T G G G C A G G G G C T C A , 3' non-translated to aa 134.
Analysis of PCR products One-third of each reaction was analyzed on a 1.6% agarose gel which was subsequently transferred to a nylon membrane by Southern blotting [29] and hybridized to an [a-32P]dCTP-oligolabelled rabbit liver cytochrome b 5 probe [19] at 65°C using Church's hybridization solution [30]. Membranes were exposed to X-ray film for 12-24 h. Some PCR products were isolated on low-melt agarose for use in reamplification reactions.
PCR amplification Amplification of 1.0 /xl of the reverse transcribed total RNA was carried out in a final volume of 50/~1 containing 200 /xM dNTPs, 0.1 /~M each of forward and reverse primers, 0.25 U / m l Taq Polymerase and buffer at a final concentration of 50 mM KC1, 10 mM Tris (pH 8.3), 1.5 mM MgC12 and 0.1% (w/v) gelatin. The mixture was overlaid with mineral oil. An MJ Research (Watertown, MA) thermal cycler was used to carry out 35 cycles of amplification consisting of denat-
A
1
2
3
4
I
I
I
I
5
Asymmetric PCR Approx. 4 ng (2 /zl) of cDNA in low melt agarose was neutralized with MgC12 and used for asymmetric PCR [32] reamplification. Conditions for PCR were those described above, except that the ratio of primers was 1:100 with the predominant primer at a final concentration of 0.1 ~zM.
6
7
8
9
I
I
I
I
10 11
I
12
13
14
15
16
I
I
I
I
I
17
18 19
I
B
C
D
Fig. 1. Agarose gel electrophoresis (a and c) and Southern blots (b and d) of cytochrome b 5 PCR products. Lane assignment is by tissue source: 1, reticulocytes; 2, leukocytes; 3, liver; 4, heart; 5, spleen; 6, pancreas; 7, ovary; 8, adrenal gland; 9, uterus; 10, femoral muscle; 11, soleus muscle; 12, bone; 13, bone marrow; 14, brain; 15, lung; 16, fat; 17, kidney; 18, gallbladder; 19, negative control. Primers were designed to amplify both the membrane-bound and the soluble forms of cytochrome b 5 mRNA (a and b), or the soluble form (c and d) only. In (a) and (b), a 203 bp product, corresponding to membrane-bound cytochrome b_s cDNA, appears in all of the tissues specimens. A slightly larger, 231 bp product, representing soluble cytochrome bs, is visualized in reticulocytes, adrenal gland, bone marrow, lung and gallbladder. The high molecular weight product seen in lanes 1, 8, 13, 15 and 18 in (a) and (b) is a PCR artifact which yields the lower molecular weight products upon reamplification. In (c) and (d), the presence of soluble b_s mRNA is confirmed in reticulocytes, adrenal gland, bone marrow, lung and gallbladder.
98
Sequencing The products of asymmetric PCR were precipitated with one-forth volume of 10 M ammonium acetate and an equal volume of isopropanol. The resultant pellets were washed twice in 70% ethanol and dried under vacuum. Direct sequencing was carried out by the dideoxy chain termination method of Sanger [33] using the single-stranded DNA from asymmetric PCR as template. Results
PCR determination of cytochrome b5 mRNA distribution A total of 18 tissues were initially selected for analysis. These included erythrocytes, leukocytes, liver, heart, spleen, pancreas, ovary, adrenal gland, uterus, fasttwitch muscle, slow-twitch muscle, bone, bone marrow, brain, lung, fat, kidney and gallbladder. Total RNA was isolated from each tissue sample, reverse-transcribed and used directly in PCR reactions. The availability of rabbit b5 genomic sequence (unpublished) and mRNA sequences for human liver and reticulocyte bss [20,22] allowed us to speculate that the mRNAs for soluble and membrane-bound rabbit bss would be homologous except for the inclusion of an additional, internal exon in the soluble b 5 mRNA. Based on the published sequence of rabbit liver cytochrome b5 m R N A [19], primer 1 (forward) and primer 5 (reverse) were designed to amplify the coding region of either mRNA. Following 35 cycles of PCR amplification the products were analyzed on agarose gels. Many of the samples appeared as smears rather than as discrete bands. This was not a totally unexpected finding, since b 5 is a member of a bs-like superfamily of proteins which includes sulfite oxidase, flavocytochrome b 2, nitrate reductase, and outer mitochondrial b5 [34]. All of these cytochromes exhibit approx. 30 to 80 percent homology to microsomal b5 in their catalytic domains and may, therefore, compete nonspecifically with b5 for primer binding. In order to increase target specificity, primer 2 (forward) and primer 4 (reverse) were constructed to amplify a smaller segment of the coding region. As seen in Fig. la, use of these primers for PCR amplification resulted in product formation from all of the tissue cDNAs. In addition, a product of larger size was apparent in five of the samples. The gel was transferred by Southern blotting and hybridized under stringent conditions to a radiolabelled rabbit liver b 5 cDNA probe. The results, as shown in Fig. lb, indicate that the products were specific for cytochrome b~. The larger-sized PCR product was believed to correspond to the putative soluble bs. However, the possibility existed that it represented a new cytochrome b5 sequence. To clarify the results, primer 3 was designed. Analysis of rabbit genomic sequence (unpublished)
primer i GAG ATG ~CT GC~ CAG TCA GAC AAA GAC GTG AAG TAC TAC ACC CTA met ala ala gln ser asp lys asp val lys tyr tyr thr leu
(14)
GAA GAG ATT AAG AAG CAC AAC CAC AGC AAA AGC ACC TGG CTG ATC glu glu ile lys lys his asn his s e r l y s s e r thr trp leu ile
(29)
CTG CAC CAC AAG GTG TAC GAT leu his his lys val try asp pr~m~ 2 CCT GGA GGG GAG GAA GTC CTG pro gly gly glu glu val leu
CTG ACC AAA TTT CTG GAG GAG CAC leu thr lys phe leu glu glu his
(44)
AGG GAA CAA GCT GGG GGC GAT GCC arg glu gln ala gly gly asp ala
(59)
ACT GAA AAC TTT GAG GAC GTC GGG CAC TCG ACA GAT GCC AGA GAG thr glu asn phe glu asp val gly his ser thr asp ala arg glu
(74)
CTG TCC AAG ACC TTC ATC ATC GGG GAG CTG CAC CCG GAT GAC AGA leu ser lys thr phe ile ile gly glu leu his pro asp asp arg
(89)
TCA AAA TTG AGC AAG CCT ATG ser lys leu s e r lys pro met
(96)
GAA ACT CTT glu thr leu pr~m~r ~ AAC TGG GTG asn trp val
IGAA C~T T A A A G G C T G T G T T T C ~ A G I {glu pro stop [ 9 8 } I
ATC ACC ACC GTC GAT TCC AAT TCC AGC TGG TGG ACC ile thr thr val asp ser asn ser ser trp trp thr (iii)
ATC CCC GCC ATC TCC GCC CTG ATC GTG GCA CTG ATG ile pro ala ile ser ala leu ile val ala leu met (126) pr~me~ 5 TAT CGC CTC TAC ATC CCC GAC GAC T~A GCCCCT~CCCAGCACCC (134) tyr arg leu try met ala asp asp stop
Fig. 2. Nucleotide sequence and deduced amino acid sequences for the m e m b r a n e - b o u n d b 5 c D N A from brain and soluble b5 c D N A from reticulocytes. Regions of homology to the oligonucleotide primers are underlined. The two sequences are identical except for the inclusion of an additional 24 bp exon (in bold type and brackets) in the reticulocyte sequence.
suggested a potential exon for soluble b5 mRNA. A sequence immediately 3' to the proposed exon was selected for use as primer 3. Fig. lc shows the results of the PCR amplification using primer 2 and primer 3. Cytochrome b5 specificity was confirmed by hybridization of a Southern blot. In addition, DNA sequencing established that all five products in Fig. lc corresponded to the soluble form of b 5.
Determination of the complete cytochrome b5 sequences The PCR products contained about 200 of the 400 or more possible bp of coding information. In order to obtain maximal sequence information, brain and bone marrow specimens were subjected to PCR amplification using primers 1 and 4 to generate the 5' coding region, or primers 2 and 5 to generate the 3' coding region. The products were isolated on low melt agarose and used as seed for asymmetric PCR reamplification reactions. The resultant single-stranded cDNAs were directly sequenced. The composite sequences from the brain and bone marrow cDNAs are shown in Fig. 2. Discussion
The results presented here demonstrate that rabbit soluble cytochrome b 5 is encoded by a mRNA that is homologous to liver b.~ mRNA, but which differs in length by 24 bp due to the inclusion of an internal exon. This 24 bp sequence is the same as that described by Takematsu et al. [35] although they had misaligned their sequence by 3 bp, because they did
99 not have the genomic sequence to specifically localize the exon. Although soluble b 5 m R N A is longer than membrane-bound b 5 mRNA, it encodes a smaller protein as a result of a termination codon in the additional exon. Our data suggest that both membrane-bound and soluble b 5 mRNAs are derived from a common precursor m R N A by alternative splicing mechanisms in a manner analagous to that shown for human b 5 [22]. The sequence of the extra 24 bp found in the soluble b5 m R N A differed from the sequence previously obtained from rabbit genomic DNA. The PCR primer 3, derived from this genomic sequence, functioned in a specific manner even though it contained five misalligned nucleotides. It has been shown previously that only the first three 5' nucleotides are absolutely critical for primer specificity, and that the primer will bind as long as there is sufficient homology in the rest of the primer sequence [36]. Based on our results, we conclude that the tissue distribution of membrane-bound cytochrome b 5 mRNA is probably ubiquitous (Fig. la,b). Because adult, skeletal muscle was previously shown to lack bs, we initially selected two muscles, the soleus and the femoral, for testing. As shown in Fig. la,b (lanes 10, 11), both samples displayed b5 mRNA. We confirmed our findings by harvesting and testing RNA isolated from six additional, different muscle specimens. All of the samples exhibited the membrane-bound b5 m R N A (results not shown). Although it could be argued that the mRNA that we detected was from leukocytes in the capillaries of the muscles, the absence of soluble b5 PCR product negates the possibility since reticulocytes are present in equal or greater number than leukocytes in whole blood. Our conclusions regarding muscle b5 are not necessarily in opposition to previous findings [23], but rather, reflect the high degree of sensitivity that can be attained with PCR techniques. We did not expect to find membrane-bound b 5 m R N A in reticulocytes. The presence of both forms of b 5 m R N A in these cells suggests that both post-transcriptional and post-translational processing are involved in the formation of soluble bs. An alternative explanation for the finding of membrane-bound b5 mRNA in reticulocytes is that the erythrocyte samples used in this study contained leukocytes. Although stained smears of the erythrocyte suspensions provided no evidence for contamination by white ceils, the sensitivity of PCR is such that the presence of one leukocyte in the sample would result in a highly visible product corresponding to membrane-bound b 5. Prior to this study, the presence of soluble cytochrome b 5 had been confirmed in erythrocytes, and suggested in kidney. The present findings (Fig. lc,d) reaffirm the presence of soluble b5 mRNA in reticulocytes and in bone marrow, the tissue in which reticulocytes are produced. The additional detection of solu-
ble b 5 m R N A in the lung, gallbladder and adrenal gland was an unexpected finding, since the soluble form of the protein was thought to function exclusively in methemoglobin reduction. These results suggest that the protein may also participate in cytoplasmic redox reactions which have not yet been elucidated. Although it could be argued that the finding of soluble b5 in the lung, gallbladder and adrenal gland is due to contamination of the tissues with reticulocytes, the absence of soluble b~ in the highly-vascularized spleen, liver and kidney refutes the argument. Considering the inherent sensitivity of PCR, we do not believe that soluble b~ is present in kidney, but we cannot exclude the possibility that tissue distribution differs in the rabbit and the pig. The use of primers 2 and 3 (Fig. lc,d) for the specific amplification of the reticulocyte b~ sequence gave results consistant with those shown in Fig. la,b. The increased levels of primer dimer product visible in Fig. ld is probably due to primer 3 not being a perfect match with the actual reticulocyte specific b~ sequence. DNA sequencing confirmed that the major signals in Fig. ld corresponded to soluble cytochrome b5 cDNA. We were unable to confirm the presence of a new, homologous b~ in brain tissue, even though the methods employed in this study allowed us to detect both the soluble and membrane-bound cytochrome b~ mRNAs. We chose to directly sequence the PCR product in order to detect any differences in m R N A sequence which might correspond to a new b 5. The nucleotide sequence of brain b 5 was identical to that of liver b~. It is unlikely that our detection of this form was due to contamination by blood pooled in capillaries because no reticulocyte form was observed. Our results suggest that the brain contains only one form of membranebound cytochrome b~ and that it is identical to that found in liver. However, the apparent conflict between our findings and the potentiometric evidence for additional forms of b~ in brain microsomes [25], may reflect changes in the protein due to post-translational processing of membrane-bound cytochrome bs.
Acknowledgement This work was supported by a grant from the National American Heart Association and its Ohio affiliate.
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