Rabbit Reticulocyte Lysate Cell-free System Research Articles

Antiproliferative and Antimitogenic Activities in a Peptide from Puffball Mushroom Calvatia caelata

A peptide with a molecular weight of 8 kDa and an N-terminal sequence closely resembling that of ubiquitin was isolated from fruiting bodies of the mosaic puffball mushroom Calvatia caelata. The peptide was purified using a protocol that involved ion-exchange chromatography on DEAE–cellulose, affinity chromatography on Affi-gel blue gel, and ion-exchange chromatography on Mono S. The peptide inhibited translation in the cell-free rabbit reticulocyte lysate system and exhibited N-glycosidase activity. It potently inhibited proliferation of spleen cells with an IC50 of about 100 nM as indicated by the suppression of [methyl-3H]thymidine uptake. The viability of breast cancer cells was reduced to half at a ubiquitin concentration of about 100 nM.

Isolation of Pleuturegin, a Novel Ribosome-Inactivating Protein from Fresh Sclerotia of the Edible Mushroom Pleurotus tuber-regium

A ribosome-inactivating protein (RIP) designated pleuturegin, which inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 0.5 nM, was purified from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Pleuturegin was distinguished from most plant and previously reported mushroom RIPs in that it was adsorbed on DEAE-cellulose and unadsorbed on SP-Sepharose, although all of them were adsorbed on Affi-gel blue gel. Pleuturegin demonstrated an N-terminal sequence that was different from those of RIPs from Flammulina velutipes (flammulin and velutin), Hypsizygus marmoreus (hypsin), and Lyophyllum shimeji (lyophyllin), the only mushroom RIPs with known N-terminal sequences. The molecular mass of pleuturegin was 38 kDa, similar to that of flammulin (40 kDa) but considerably larger than those of velutin (13.8 kDa), hypsin (20 kDa), and lyophyllin (20 kDa). Pleuturegin was devoid of ribonuclease activity.

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation.

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of β- and γ-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P < 0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P < 0.05). The β- and γ-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.

The 5'-untranslated region of GM-CSF mRNA suppresses translational repression mediated by the 3' adenosine-uridine-rich element and the poly(A) tail

Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA levels are controlled post-transcriptionally by the 3'-untranslated region (UTR) adenosine-uridine-rich element (ARE). In untransformed, resting cells, the ARE targets GM-CSF mRNA for rapid degradation, thereby significantly suppressing protein expression. We used a rabbit reticulocyte lysate (RRL) cell-free system to examine translational regulation of GM-CSF expression. We uncoupled decay rates from rates of translation by programming the RRL with an excess of mRNAs. Capped, full-length, polyadenyl-ated human GM-CSF mRNA (full-length 5'-UTR AUUUA+A90) and an ARE-modified version (full-length 5'-UTR AUGUA+A90) produced identical amounts of protein. When the 5'-UTR was replaced with an irrelevant synthetic leader sequence (syn 5'-UTR), translation of syn 5'-UTR AUUUA+A90 mRNA was suppressed by >20-fold. Mutation of the ARE or removal of the poly(A) tail relieved this inhibition. Thus, in the absence of a native 5'-UTR, the ARE and poly(A) tail act in concert to block GM-CSF mRNA translation. Substitutions of different regions of the native 5'-UTR revealed that the entire sequence was essential in maintaining the highest rates of translation. However, shorter 10-12 nt contiguous 5'-UTR regions supported 50-60% of maximum translation. The 5'-UTR is highly conserved, suggesting similar regulation in multiple species and in these studies was the dominant element regulating GM-CSF mRNA translation, overriding the inhibitory effects of the ARE and the poly(A) tail.

In vitro translation of messenger RNA in a rabbit reticulocyte lysate cell-free system.

The identification of specific messenger RNA molecules and the characterization of the proteins encoded by them, has been greatly assisted by the development of in vitro translation systems. These cell-free extracts comprise the cellular components necessary for protein synthesis, i.e., ribosomes, tRNA, rRNA, amino acids, initiation, elongation and termination factors, and the energy-generating system (1). Heterologous mRNAs are faithfully and efficiently translated in extracts of HeLa cells (2), Krebs II ascites tumor cells (2), mouse L cells (2), rat and mouse liver cells (3), Chinese hamster ovary (CHO) cells (2), and rabbit reticulocyte lysates (2,4), in addition to those of rye embryo (5) and wheat germ (6). Translation in cell-free systems is simpler and more rapid (60 min vs 24 h) than the in vivo translation system using Xenopus oocytes.

Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems

The Ure2p yeast prion-like protein was translated in vitro in the presence of labeled [ 35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31–34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93–95]. Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.

Translational control of poly(A)-binding protein expression.

Poly(A)-binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract. In many eukaryotic cells, this mRNA is inefficiently translated. The 5' untranslated region (UTR) of PABP mRNA has several adenine-rich regions which may serve as the PABP-binding sites to control its translation by a feed-back mechanism. This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell-free system. Results of our studies show that removal of the putative PABP-binding sites from the 5' UTR of this mRNA enhances its translation in the rabbit reticulocyte cell-free system. Furthermore, in vitro translation of the full-length PABP mRNA was inhibited by addition of purified PABP to the cell-free system. In contrast, translation of truncated mRNA lacking the putative PABP-binding sites at the 5' UTR was not inhibited by exogenous PABP. We have also tested the ability of purified PABP to bind to the 5' UTR of PABP mRNA using ultraviolet-mediated covalent cross-linking of RNA and proteins in vitro. Our results show that exogenous PABP binds to the 5' UTR of its full-length mRNA. Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5' UTR. The adenine-rich regions are located within this segment of PABP mRNA. Following incubation of PABP mRNA in the reticulocyte lysate cell-free system under conditions of mRNA translation, the polysomal and non-translated free mRNA fractions were separated by centrifugation. Analysis of free and polysomal mRNA-protein (mRNP) complexes following ultraviolet-induced cross-linking showed that the free mRNP population was preferentially enriched in PABP. Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed-back mechanism.

The Amino Acid at the X Position of an Asn-X-Ser Sequon Is an Important Determinant of N-Linked Core-glycosylation Efficiency

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.

In vitro translation of messenger RNA in a rabbit reticulocyte lysate cell-free system.

The identification of specific messenger RNA molecules and the characterization of the proteins encoded by them, has been greatly assisted by the development of in vitro translation systems. These cell-free extracts comprise the cellular components necessary for protein synthesis, i.e., ribosomes, tRNA, rRNA, amino acids, initiation, elongation and termination factors, and the energy-generating system (1). Heterologous mRNAs are faithfully and efficiently translated in extracts of HeLa cells (2), Krebs II ascites tumor cells (2), mouse L cells (2), rat and mouse liver cells (3), Chinese hamster ovary (CHO) cells (2), and rabbit reticulocyte lysates (2,4), in addition to those of rye embryo (5) and wheat germ (6). Translation in cell-free systems is simpler and more rapid (60 min vs 24 h) than the in vivo translation system using Xenopus oocytes.

Kinetics of translation of γB crystallin and its circularly permutated variant in an in vitro cell-free system: possible relations to codon distribution and protein folding

Analysis of nascent γB-crystallin peptides accumulating during in vitro translation in a rabbit reticulocyte lysate cell-free system was carried out. As a consequence of the irregular distribution of rare codons along the polypeptide chain of γB-crystallin, translation of the two-domain protein is a non-uniform process characterized by specific pauses. One of the major delays occurs during the translation of the connecting peptide between the domains. Comparing the kinetics of translation of natural γB-crystallin and its circularly permutated variant (with the order of the N- and C-terminal domains exchanged) reveals that the natural N-terminal domain is translated faster than the C-terminal one. Since the N-terminal domain in natural γB-crystallin is known to be more stable and to fold faster than the C-terminal one [E.-M. Mayr et al. (1994) J. Mol. Biol. 235, 84–88], the present data suggest that the translation rates are optimized to tune the synthesis and folding of the nascent polypeptide chain. In this connection, the pause in the linker region between the domains provides a delay allowing the correct folding of the N-terminal domain and its subsequent assistance in the stabilization of the C-terminal one.

An improved method for extraction of mRNA and protein from the fungus Chaetomium gracile with anhydrous hydrogen fluoride.

We previously reported that DNAs directly applicable to restriction analyses and transformation of Escherichia coli can be extracted from fungi and yeasts by use of anhydrous hydrogen fluoride (HF) under a mild condition, 5 min at 0 degrees C [Oshiro, S., Katsura, N., Kitada, K., and Gunge, N. (1987) FEBS Lett. 220, 383-386]. In the present investigation, we examined whether this improved method is also applicable to extraction of RNA and protein from the fungus Chaetomium gracile. The RNA and protein were effectively extracted from the fungus after anhydrous hydrogen fluoride (HF) treatment for a short time (1 min) at 0 degrees C. The extracted poly(A)-enriched mRNA and proteins were fully intact: the mRNA purified by messenger-activated paper with poly(U) directed not only the incorporation of [3H]glycine into polypeptides but also the synthesis in a rabbit reticulocyte lysate cell-free system of proteins reactive to antibodies against the soluble fraction extracted from the fungus by HF. Analyses by gel filtration and polyacrylamide electrophoresis under native conditions showed that dextranase extracted from the fungus by HF under the same conditions had the same molecular weight and electrophretic mobility as the enzyme excreted into the medium. This suggests that the mRNAs and proteins extracted by this method are also applicable to protein synthesis directed in a cell-free system and to enzyme purification from a fungus insusceptible to lytic enzymes. This method provides a pure preparation of mRNA within 5 h and starting materials for protein purification within 1 h.

The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting.

The genomic RNA of human astrovirus serotype 1 (HAst-1) contains three open reading frames (ORFs), 1a, 1b, and 2. ORF 1b is located downstream of, and overlaps, 1a, and it has been suggested on the basis of sequence analysis that expression of ORF 1b is mediated through -1 ribosomal frameshifting. To examine this possibility, a cDNA fragment containing the 1a-1b overlap region was cloned within a reporter gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Synthetic transcripts derived from this plasmid, when translated in the rabbit reticulocyte lysate cell-free system, specified the synthesis of polypeptides whose size and antibody reactivity were consistent with an efficient -1 ribosomal frameshift event at the overlap region. The HAst-1 frameshift signal has two essential components, a heptanucleotide slippery sequence, A6C, and a stem-loop structure in the RNA. The presence of this structure was confirmed by complementary and compensatory mutation analysis and by direct structure probing with single- and double-stranded RNA-specific reagents. The HAst-1 frameshift signal, like that present at the overlap of the gag and pro genes of the retrovirus human T-cell lymphotrophic virus type II, does not involve the formation of an RNA pseudoknot.

Identification of important residues within the putative nucleoside binding site of HSV-1 thymidine kinase by random sequence selection: analysis of selected mutants in vitro.

Random sequence mutagenesis in conjunction with genetic complementation was used to map the function of amino acid residues within the putative nucleoside binding site of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). Six codons of the putative nucleoside binding site of the HSV-1 tk were substituted by a duplex of extended oligonucleotides containing 20% random sequences. Approximately 260 mutants were screened for the ability to genetically complement a TK-deficient Escherichia coli. Of those screened, 32% conferred TK activity. Approximately 60% of the TK positive clones contained single amino acid changes, 23% contained double changes, and 13.4% encoded the wild-type TK amino acid sequence. A small percentage of clones, 2.4% and 1.2%, contained triple or quadruple alterations, respectively. Three residues (D162, H163, and R164) appeared to be highly conserved especially with regard to the type of residues able to substitute. Secondary screening results indicated that several of the mutants had higher affinities for acyclovir and/or 3'-azido-3'-deoxythymidine than thymidine in complementation assays. In addition, a number of clones were unable to form colonies on selection medium at elevated temperatures (42 degrees C). Eight selected mutants were subcloned into an in vitro transcription vector and the derived transcripts used to program a rabbit reticulocyte lysate cell-free translation system. Biologically active translation products were then analyzed in vitro for thymidine kinase activity, for thermal stability, and for the ability to phosphorylate selected nucleoside analogues. Two of the eight mutants had an elevated thymidine kinase activity, two were significantly thermolabile, and three exhibited enhanced efficiency in phosphorylation of nucleoside analogues.

Inherited congenital adrenal hyperplasia in the rabbit: absent cholesterol side-chain cleavage cytochrome P450 gene expression.

We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in vitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 +/- 5 mg/adrenal; normal, 2.7 +/- 1.0 mg/adrenal). Serum pregnenolone (delta 5-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 +/- 0.05 microgram/dl; P less than 0.001 vs. normal (0.66 +/- 0.57)]. Serum Na+ levels in CAH newborn rabbits were in the normal range (143 +/- 30 meq/liter), but K+ levels were elevated [7 +/- 1.1 meq/liter; P less than 0.05 vs. normal (5.9 +/- 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [3H] cholesterol (30-100 pmol/flask) and ACTH (100 mU/flask) produced [3H]delta 5-P (newborn, 21 and 45 fmol/100 mg; adult, 3 and 5 fmol/100 mg) and [3H]corticosterone (newborn, 23 fmol/100 mg; adult, 11.3 fmol/100 mg), but CAH adrenals produced no product (less than 1.3 fmol/100 mg). Adrenal mitochondria from normal newborn rabbits produced delta 5-P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11 beta-hydroxylase activity. A Western blot of adrenal homogenate from normal newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x 10(3), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17 alpha-hydroxylase cytochrome P450 (P450(17)alpha) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [35S] methionine yielded a precursor P450scc protein (mol wt, 58.5 x 10(3)) with normal adrenal RNA, but not with CAH adrenal RNA. P450scc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21-Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P450scc gene expression. The clinical, pathological, and biochemical manifestations of P450scc deficiency in the rabbit are nearly identical to the human disorder. Increased 11 beta-hydroxylase activity and increased P450(17)alpha on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P450scc gene in this animal CAH model will facilitate understanding of P450scc deficiency CAH.

Characterization of the roles of conserved cysteine and histidine residues in poliovirus 2A protease

The primary processing of the poliovirus polyprotein is catalyzed by 2A protease (2A pro) which cleaves at the 1D/2A junction in a very rapid cotranslational reaction. In addition, 2A pro also indirectly induces cleavage of the p220 component of eIF-4F, which results in selective inhibition of host protein synthesis. Earlier studies have indicated that 2A pro is related to 3C protease (3C pro) and is structurally similar to trypsin-like serine proteases with the substitution of Cysl09 as the nucleophile. We noticed that 2A pro of enteroviruses and rhinoviruses contains a specific motif of Cys55-XaaCys57-Xaa n-Cysl15-Xaa-His117 which is absolutely conserved, but which is not found in viral 3C pro or known cellular serine proteases. To better understand the specific roles these conserved cysteine and histidine residues played in the structure/function of 2A pro, we constructed a series of 2A pro mutants by site-specific mutagenesis and analyzed the mutant enzymes with respect to their biochemical properties. Conservative amino acid replacements at Cys55, Cys57, Cys115, or His117 resulted, in each case, in a complete loss of both in cis and in trans activities of 2A pro. To determine the function of these residues, we examined the biochemical/structural features of 2A pro expressed in a cell-free rabbit reticulocyte lysate system. Gel mobility shift and chemical modification data suggest that these cysteine residues do not form intra-molecular disulfide linkages as a structural feature of 2A pro. However, studies with metal chelators did not eliminate the possibility that 2A pro contains a metal-binding ligand. Finally, our results suggest that these conserved cysteine and histidine residues, including Cys55, Cys57, Cysl15, and Hisl17, are critical in maintaining the active conformation of 2A pro structure and essential in supporting the catalytic activity of 2A pro.

Characterization and molecular properties of 2-oxoglutarate decarboxylase from Euglena gracilis

2-Oxoglutarate decarboxylase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. It had a molecular weight of 250,000 and consisted of four identical subunits of molecular weight 62,000. The enzyme was specific for 2-oxoglutarate, but not for other 2-oxo acids such as pyruvate and oxalacetate. Thiamin pyrophosphate and MgCl 2 were required for maximum activity. The K m values of the enzyme for 2-oxoglutarate, thiamin pyrophosphate, and MgCl 2 were 330, 56, and 93 μ m, respectively. 2-Mercaptoethanol and NADP + augmented significantly the enzyme activity. The amino acid composition and amino acid sequence of the amino-terminal region of 2-oxoglutarate decarboxylase were determined. On Ouchterlony double-immunodiffusion gels, the anti-2-oxoglutarate decarboxylase antibody gave sharp precipitin lines against the mitochondrial fraction of E. gracilis and the purified 2-oxoglutarate decarboxylase, but not against pyruvate decarboxylase from Saccharomyces cerevisiae. On Immunoblots of the crude extract of Euglena, the antibody recognized two polypeptides whose molecular weights were 62,000 and 65,000, respectively. The polypeptide with the molecular weight of 62,000 was found only in mitochondrial fractions. In vitro translation of Euglena polyadenylated RNA in a cell-free rabbit reticulocyte lysate system explained the formation of a single polypeptide with a molecular weight of 65,000, suggesting that a putative precursor of 2-oxoglutarate decarboxylase which is about 3000 larger than the subunit of the mature enzyme is synthesized in Euglena cells.

Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

Transfected NA1 and NA2 Forms of Human Neutrophil Fc Receptor III Exhibit Antigenic and Structural Heterogeneity

Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.