Import of chemically synthesized signal peptides into rat liver mitochondria

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.     

Liver mitochondrial aldehyde dehydrogenase: in vitro expression, in vitro import, and effect of alcohols on import

An in vitro expression plasmid (pGRAP) that contained the cDNA coding for the rat mitochondrial aldehyde dehydrogenase precursor was constructed, mRNA was synthesized then translated, and the in vitro synthesized precursor of aldehyde dehydrogenase was used in an in vitro import assay. As expected the 19 amino acid signal peptide of the precursor allowed import of the precursor into rat liver mitochondria. This in vitro system was used to examine the effect of alcohols on import. It was found that the alcohols (ethyl, butyl, hexyl, and octyl) tested inhibited the import of the aldehyde dehydrogenase precursor. Pretreatment of the mitochondria with alcohol was responsible for the inhibition. The inhibition appeared to be relatively specific for pre-aldehyde dehydrogenase as the precursor of ornithine transcarbamylase was still imported in the presence of alcohols. Of potential physiological significance was finding that ethanol inhibited import in a dose-response fashion; 50% inhibition occurred at 75 mM, a concentration achievable during the ingestion of alcohol. In addition, the concentrations of alcohols required to produce an inhibitory effect on import decreased as the hydrocarbon chain length of alcohols increased. The inhibitory effect of alcohols appeared to be specific as other solvents examined did not inhibit import. We postulate that alcohols may perturb the mitochondrial membrane and affect the receptor-translocator necessary for the import of the aldehyde dehydrogenase precursor.     

Recognition of rat liver and kidney nuclear T3 receptors by an antibody against c-erb A peptide

It has been reported that c-erb A encodes nuclear T3 receptors (NT3R). Based on the sequence of c-erb A cDNA, we synthesized a polypeptide consisting of 15 amino acids, the sequence of which has high homology between c-erb A alpha 1 and beta. The antibody against this c-erb A peptide not only immunoprecipitated rat liver and kidney NT3R but also inhibited T3 binding to NT3R. In a displacement study, the inhibition of [125I]T3-binding by the antibody was parallel to that by T3 in terms of the concentration of the competitor added in the incubation mixture. Scatchard analysis revealed that the antibody decreased the value for the association constant in a dose dependent manner. The antibody did not bind T3 itself. The results show that the antibody against c-erb A peptide recognizes rat liver and kidney NT3R and that the sequence encoding this peptide, the closest carboxyl-terminal of c-erb A may be critical or at least closely related to the hormone binding.     

Role of heat shock cognate 70 protein in import of ornithine transcarbamylase precursor into mammalian mitochondria.

The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.     

Development and characterization of cholinephosphotransferase antibody

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.     

Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein

An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.     

Production of anti-rat islet cell surface antibody in guinea pigs

Anti-rat islet serum was prepared in guinea pigs by multiple subcutaneous inoculations of rat islets homogenates emulsified in complete Freund's adjuvant (CFA). The anti-rat islet serum was cytotoxic against rat spleen cells in the presence of complement and the nonspecific antibodies were observed with homogenates of rat livers and spleens. After absorption, the serum lost the cytotoxicity against the rat spleen cells yet showed specific cytotoxicity against the rat islet cells. The binding capacity of anti-rat islet antibody was determined by the indirect immunofluorescence test using FITC conjugated rabbit anti-guinea pig IgG serum. As the guinea pig anti-rat islet serum contained anti-insulin antibody, the role of this antibody in this cytotoxic activity and surface immunofluorescence was studied. However, the anti-insulin antibody used as the control showed neither cytotoxicity nor surface immunofluorescence. After neutralizing the anti-insulin antibody in the antiserum with insulin, the serum remained cytotoxic to the rat islet cells and a surface immunofluorescence appeared. These data show that specific anti-rat islet cell surface antibody can be produced in guinea pigs by multiple inoculations of rat islets homogenates with CFA.     

cDNA cloning and sequencing for the import precursor of coupling factor 6 in H(+)-ATP synthase from rat liver mitochondria

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

On the organ specificity of neutral glycerolester hydrolase of various tissues; an immunological study

Rat liver microsomal glycerol monoester hydrolase (EC 3.1.1.23) has been purified 130 fold. The enzyme has a molecular weight of about 60,000. An antibody raised against this enzyme in rabbit did not inhibit heparin-releasable liver lipase, which hydrolyses long-chain 1- and 2-monoglycerides effectively. This confirms an earlier conclusion, based on results obtained with an antibody raised against the latter enzyme, that the non-releasable and heparin-releasable liver enzymes are different proteins. The antibody against the liver microsomal glycerol monoester hydrolase, however, inhibited also the monoglyceridase activities of acetone powder extracts of rat small intestinal epithelial microsomes and rat epididymal fat pads, suggesting structural similarities between the endoplasmic reticulum hydrolases of various tissues. These findings also apply to pig where an antibody against adipose tissue lipases inhibits the monoglyceridase activities of small intestinal and liver microsomal acetone powder extracts.     

Immuno-cross-reactivity of histidine and dopa decarboxylases

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

Cellular import of synthetic peptide using a cell-permeable sequence in plant protoplasts

In this paper, we describe a rapid method to incorporate biologically active synthetic peptide in plant protoplasts. The peptides used contain a hydrophobic membrane permeable sequence as a carrier for the import through the plasma membrane. The membrane permeable sequence corresponds to the h-region, the more hydrophobic domain found in the signal peptide of secreted proteins. To evaluate the feasibility of the method, we synthesized a cell-permeable peptide with an h-region of a plant signal peptide plus residues 410–419 of the human c-myc oncogene product. Detection was performed via fluorescence analysis using specific monoclonal anti-c-myc primary antibody and FITC-conjugated secondary antibody. No saturation of import was observed, suggesting that the mechanisms involved do not require energy. The half-life time of the internalized peptide was estimated and results indicate that peptide concentration into protoplasts was constant for 8 h following incorporation. This method is complementary to microinjection or to the use of membrane permeabilizing reagents to study in vivo protein–protein or DNA–protein interactions. Finally, this method was used to analyse a putative interaction between the conserved cytoplasmic tail of a transmembrane receptor (HaELP, Helianthus annuus EGF receptor like protein) and the cytoskeleton. No interaction was found between these components.     

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

A monoclonal antibody against human kidney gamma-glutamyl transpeptidase: preparation, immunochemical, and immunohistochemical characterization

To perform immunohistochemical study of the distribution of gamma-glutamyl transpeptidase in human organs, a highly specific antibody against the human enzyme is required. We prepared monoclonal antibody against gamma-glutamyl transpeptidase from human kidney, using the hybridoma technique. The antibody was of the IgG1 type and the light chain belonged to the kappa class. The antibody reacted specifically with the 63 KD heavy subunit of the enzyme. Examination of the specificity of the antibody performed by immunohistochemical staining of human kidney sections revealed that the antigen was localized on the brush border and along the basolateral membrane of the epithelial cells of both the convoluted and the straight portions of the proximal tubule. This antibody was also reactive in several human organs other than kidney, including epididymis, prostate, seminal vesicle, pancreas, and normal liver, and in human hepatoma. These findings indicate the existence of an antigenic determinant common to human kidney and other organs. The monoclonal antibody did not crossreact with mouse, rat, guinea pig, rabbit, or pig kidney.     

Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors.     

Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli

The cDNA coding for the precursor (p-ALDH) or mature (m-ALDH) rat liver mitochondrial aldehyde dehydrogenase was cloned in an expression vector pT7-7 and expressed in Escherichia coli strain BL21 (DE3)/plysS. The p-ALDH expressed in E. coli was a soluble tetrameric protein. It exhibited virtually the same specific activity and KmS for substrates as m-ALDH. N-terminal sequencing of isolated p-ALDH provided the evidence that the catalytic activity was not derived from a partially processed mature-like enzyme. The assembly states of both p-ALDH and m-ALDH synthesized in a rabbit reticulocyte lysate were also determined. Both of them were monomers and could not bind to a 5'-AMP-Sepharose column, showing that the monomeric form of the enzyme is inactive. The stabilities in vivo and in vitro were compared between p-ALDH and m-ALDH expressed in E. coli. p-ALDH was less stable than was m-ALDH both in vivo and in vitro. Thus, although the conformations of p-ALDH and m-ALDH are similar, the presence of signal peptide is a destabilizing factor to the p-ALDH. p-ALDH expressed in E. coli could bind to and be translocated into rat liver mitochondria, however, with lower efficiency when compared to the import of p-ALDH synthesized in reticulocyte lysate.     

Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (AT1) receptor.

A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-1 (AT1) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [3H]-DuP753 binding and AT1 receptor mRNA.     

Design and application of a polyclonal peptide antiserum for the universal detection of leptin protein

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the coding sequence reported for the porcine leptin gene (GenBank Accession No. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied to date. Purified recombinant human, mouse, rat, pig, and chicken leptin proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The peptide antiserum was found to be highly specific for leptin which exhibited an estimated molecular weight of about 16 kDa for all species analyzed. The sensitivity of the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chicken plasma using a combination of gel filtration and ion exchange column chromatography. Slot blots indicated a potential application of the immunostaining technique for quantitative analysis of leptin protein. Finally, the peptide antiserum was successfully employed to localize leptin protein by immunohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to report a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin.