High Molecular Weight Precursor Research Articles

Detection in BHK cells of a precursor form for lamin A

Lamins are structural proteins found in the fibrous lamina underlining the nuclear envelope. In vitro translation of polyadenylated RNA or polysomes followed by immunoprecipitation with a serum raised against BHK nuclear matrix proteins showed that lamin A (72 kD) is synthesized as a high molecular weight precursor (74 kD) (Laliberté et al., J cell biol 98 (1984) 980) [23]. We have thus investigated the presence in BHK cells of this putative precursor by in vivo labelling with [ 35S]methionine and immunoprecipitation of lamin proteins. Short labelling times, ranging from 5 to 60 min reveal the presence of the 74 kD protein. Pulse-chase experiments indicate that the half-life of the precursor is about 60 min. On two-dimensional gel, the 74 kD protein is resolved in a cluster of isovariants between pH 7.4 and 6.6, which are generally slightly more alkaline than their counterparts in lamin A. These results indicate that lamin A is synthesized as a precursor of 74 kD; the long half-life further suggests that pre-lamin A might accumulate in some sort of cellular pool before undergoing post-transcriptional modification(s) to give the mature form of lamin A.

Cyclical secretion of prorenin during the menstrual cycle: synchronization with luteinizing hormone and progesterone.

Plasma prorenin, a high molecular weight precursor form of renin, (renin, EC 3.4.23.15; old number, EC 3.4.99.19), was measured three times weekly in normal young women during the menstrual cycle and was related to changes in luteinizing hormone, estradiol, and progesterone. In all subjects a stable baseline level of prorenin occurred during the follicular phase. Then, simultaneously or soon after the luteinizing hormone peak, plasma prorenin consistently increased about 2-fold. Baseline prorenin ranged from 18 to 40 ng per ml per hr, and peak prorenin ranged from 35 to 65 ng per ml per hr. The maximum increase in prorenin averaged 80%. Prorenin remained elevated during the mid-luteal phase of the menstrual cycle and returned to baseline during the late-luteal phase in coordination with the decrease in progesterone. The changes in prorenin were not synchronized with changes in active renin which was significantly increased only during the mid-luteal phase. These findings suggest that prorenin may be involved in reproductive physiology.

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals.

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.

Human cerebrospinal fluid somatostatin in neurologic disease

Concentrations of somatostatin-like immunoreactivity (SLI) were examined in human cerebrospinal fluid (CSF). To validate the assay it was shown that CSF which had been run over a somatostatin immunoaffinity column showed no interference with binding of synthetic standards. Reversed phase HPLC showed that the immunoreactive material coeluted with SS14 and SS28 as well as a higher molecular weight precursor. Concentrations of human CSF SLI were stable at both room temperature and 4 °C for up to 72 h while repeated freezing and thawing resulted in a significant loss of immunoreactive material after the 3rd repetition. In normal control patients less than 55 years of age, CSF SLI was 54.7 ± 1.9 pg/ml, while in those older than 55 CSF SLI was 56.2 ± 2.2 pg/ml. Febrile infants had significantly higher levels (75.4 ± 7.3) pg/ml. CSF SLI was normal in patients with aseptic meningitis (54.4 ± 3.4 pg/ml), suggesting that increased CSF protein and white cell counts do not affect concentrations. Concentrations of CSF SLI were significantly increased in intervertebral disc disease (65.1 ± 5.6 pg/ml), intrinsic spinal cord pathology (101.0 ± 23.9 pg/ml), central nervous system tumors (78.0 ± 7.8 pg/ml) and acute cortical damage of varied etiology (277.8 ± 81.6 pg/ml). Patients with pseudotumor cerebri had concentrations of 43.2 ± 2.5 pg/ml. Concentrations of CSF SLI were significantly reduced ( P < 0.01) in multiple sclerosis (38.8 ± 5.5 pg/ml) and old cortical pathology (23.2 ± 3.9 pg/ml). Serial CSF analyses in patients with acute CNS lesions, suggest that CSF SLI may be a neurochemical marker of acute pathology, as the initially elevated levels fell to or below normal with resolution of the pathologic process.

Changes in the processing of β-endorphin in the hypothalamus and pituitary gland of female rats during sexual maturation

Puberty in the female rat is accompanied by a marked attenuation of the opioid inhibition of luteinizing hormone secretion. One factor which may contribute to this altered role is a change in the metabolism of opioid peptides during sexual maturation. β-Endorphin undergoes a considerable degree of metabolism through both C-terminal proteolysis and N-acetylation, and these metabolites do not possess opioid activity. The processing of β-endorphin in the hypothalamus and in the anterior and neurointermediate lobes of the pituitary gland in prepubertal and adult female rats was studied using gel filtration and high performance liquid chromatography coupled with radioimmunoassay. In the anterior lobe, high molecular weight precursors of β-endorphin (pro-opiomelanocortin and β-lipotropin) were present in prepubertal (28 days old) rats, but little authentic β-endorphin was detected. In contrast, only β-lipotropin and β-endorphin were present in mature (70 days old) animals. Only β-endorphin-sized peptides were present in the neurointermediate lobes of both prepubertal and adult rats. However, the proportion of N-acetylated metabolites was higher in sexually mature animals. In the hypothalamus, only β-endorphin-sized peptides were present in both juvenile and adult animals. However, C-terminal proteolysis increased with age (no acetylated metabolites were detectable in this tissue). The proportion of the total β-endorphin-like immunoreactivity attributable to β-endorphin was lower in young adult (first dioestrus after vaginal opening) (55%) and mature (dioestrus, 61–64 days old) rats (56%) compared to prepubertal (30 days old) animals (75%) and the proportions of non-acetylated metabolites [β-endorphin-(1–27) in young adults and β-endorphin-(1–26) in adults] were increased concomitantly. These changes were correlated with a reduced luteinizing hormone response to the opiate antagonist naloxone in adult compared to prepubertal rats. β-Endorphin is processed differently in the two lobes of the pituitary gland and in the hypothalamus and the degree of metabolism increases as the rat reaches sexual maturity. The increased metabolism of β-endorphin in the hypothalamus, the site most likely to be involved in the control of luteinizing hormone secretion, results in a reduction in the relative proportion of the opioid active parent peptide. Thus, increased inactivation of β-endorphin may contribute to the attenuation of the opioid inhibition of luteinizing hormone secretion observed during puberty.

Intracellular transport of high molecular weight intermediates of acid α-glucosidase in human fibroblasts

Two transient, high molecular weight precursors of human acid α-glucosidase were detected by immune precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The high molecular weight precursors were rapidly converted into lower molecular weight forms corresponding to previously identified intermediates of acid α-glucosidase. An accumulation of these precursors was observed in fibroblasts treated with monesin and nigericin, suggesting that these precursors are intermediates of acid α-glucosidase undergoing transport through the Golgi complex.

Ser-Leu-Arg-Arg-atriopeptin III: the major circulating form of atrial peptide.

Vasopressin induces a concentration-dependent increase in atriopeptin immunoreactivity in plasma. Rat plasma, rat atrial extract, and synthetic atriopeptin III (APIII) produced parallel displacement curves of iodine-125-labeled APIII binding to specific antiserum. Fractionation of plasma atriopeptin immunoreactivity by reverse-phase high-performance liquid chromatography showed that the major portion consists of two species of low molecular weight peptides in a ratio of 10 to 1. Both peaks exhibited potent vasorelaxant activity, suggesting the presence of the carboxyl terminal Phe-Arg sequence of atriopeptin in each species. Sequence determination of the purified peptides indicated that the major peptide is Ser-Leu-Arg-Arg-APIII and the minor peptide APIII. It appears that the former is the major species of atrial peptide in the rat circulation and that it is the product of selective cleavage of the high molecular weight precursor.

Biosynthesis of rat liver transhydrogenase in vivo and in vitro.

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.

The structure and biosynthesis of epidermal growth factor precursor.

The structure of mouse submaxillary gland epidermal growth factor (EGF) precursor has been deduced from complementary DNAs. The mRNA is approximately 4800 bases and predicts prepro EGF to be a protein of 1217 amino acid residues (133 X 10 Mr). EGF (53 amino acid residues) is flanked by polypeptides of 188 and 976 residues at its carboxy and amino termini, respectively. The amino terminus of the precursor contains seven cysteine-rich peptides that resemble EGF. Towards the carboxy terminus is a 20-residue hydrophobic membrane spanning domain. The mild portion of the EGF precursor shares a 33% homology with the low density lipoprotein receptor, which extends over 400 amino acid residues. These features suggest that EGF precursor could function as a membrane-bound receptor. RNA dot-blot analysis and in situ hybridization show EGF mRNA to be abundant in the submaxillary gland, kidney and incisor tooth buds. Lower EGF mRNA levels were found in the lactating breast, pancreas, small intestine, ovary, spleen, lung, pituitary and liver. In the kidney EGF mRNA was most abundant in the distal convoluted tubules. Analysis of EGF precursor biosynthesis in organ culture of the submaxillary gland and kidney showed differential processing of the precursor in the two tissues. In the submaxillary gland immunoreactive low molecular weight EGF was produced, but in the kidney the high molecular weight precursor was not processed. In the distal convoluted tubule of the kidney EGF precursor may act as a receptor that is involved in ion transport.

Synthesis and intracellular processing of sucrase-isomaltase in rat jejunum

To examine directly the synthesis and intracellular processing of rat intestinal sucrase-isomaltase, [3H]leucine-labeled enzyme was solubilized from purified microsomal and microvillus fractions, immunoprecipitated, and analyzed. Immunologic identity between the microsomal and microvillus forms of the enzyme was demonstrated. Time-course studies using both intravenous and intraluminal labeling demonstrated an initial peak of microsomal sucrase-isomaltase labeling at 30 min followed by a sharp decline, and a subsequent rise in microvillus sucrase-isomaltase labeling, suggesting a precursorproduct relationship. Fluorography and quantitative analysis of electrophoresed immunoprecipitates confirmed these results, and revealed the presence of two high molecular weight proteins in the microsomes. Susceptibility of the smaller high molecular weight precursor to cleavage with endo-β-N-acetylglucosaminidase H indicated that this was the initially synthesized form of the glycoprotein. These data are consistent with the membrane flow hypothesis and the single polypeptide model suggested for sucrase-isomaltase synthesis.

Native opioid-like peptides in Squilla mantis ganglia

To detect the presence of a mammalian-like enkephalin precursor in suboesophageal ganglia of Squilla mantis, an arthropod shown to be sensitive in vivo to opiates [8], protein acid extracts were fractionated by gel filtration into three large pools: A(Mr>65,000), B(10,000<Mr<65,000) and C (Mr<10,000). Only the low molecular weight pool, pool C, showed opioid-like activity when assayed by displacing labeled D-Ala2-D-Leu5-enkephalin from rat brain membranes. After trypsin and carboxypeptidase B proteolysis, pool A remained inactive, while pool B turned out to be active and was shown to inhibit the twitch response of electrically stimulated guinea-pig ileum. After HPLC fractionation of proteolyzed pool B, most of the opioid-like activity was found to be associated with a fraction showing an elution volume different from that of opioid peptide standards. Furthermore, no fraction showed immunoreactivity with anti-Met-enkephalin antibodies. The results suggest that native opioid-like peptides are present in Squilla mantis and are most likely released from higher molecular weight precursor(s).

Enkephalins in Pheochromocytoma: Studies in a Rat Model

Enkephalins, endogenous opioid pentapeptides which are found in high concentration in normal chromaffin tissue, may play a role in blood pressure regulation. We therefore examined the presence and actions of enkephalins in pheochromocytoma in a rat model. Transplantable norepinephrine-rich tumors, which gave rise to significant blood pressure elevations, contained measurable immunoreactive enkephalins as determined by specific radioimmunoassays for leucine-enkephalin and methionine-enkephalin. Enkephalin immunoreactivity paralleled the enkephalin assay standard curves and was not abolished by boiling or by protease inhibitors (EDTA, PMSF). Authenticity of the immunoreactive enkephalins was confirmed by reverse-phase high pressure liquid chromatography. The amount of enkephalin immunoreactivity present initially in these tumors was greatly augmented by the prohormone activators trypsin or trypsin plus carboxypeptidase B, suggesting that most of the immunoreactive enkephalin was present in higher molecular weight precursor form. Enkephalin determinations on human pheochromocytoma and catecholamine measurements in both rat and human pheochromocytoma, demonstrated certain similarities and differences in enkephalin and catecholamine content between rat and human tumors. Total tumor enkephalins correlated (r = 0.91, p less than 0.05) with total tumor catecholamines in rat pheochromocytoma, suggesting co-regulation of synthesis of these 2 chromaffin tissue substances. Physiologic studies, in which intravenous leucine-enkephalin and the opioid antagonist nalaxone were administered to pheochromocytoma-implanted rats and sham-operated controls, failed to uncover an opioid peptide influence upon blood pressure in this animal model of pheochromocytoma.

Proteolytic cleavage and maturation of the crystalline secretion products of Paramecium

The secretory vesicles (trichocysts) of the unicellular eukaryote Paramecium provide a model system for genetic, cytological and biochemical studies of secretory processes. An additional interest in trichocysts lies in the crystalline organization of their content, before and after exocytosis. We have analysed the biosynthesis of the secreted proteins and the building up of their crystalline packing by a combination of methods using: (1) antibodies raised against the secreted products; (2) mutants blocked at different steps of the secretory pathway; and (3) the carboxylic ionophore monensin. Our results support the following conclusions: firstly, the secreted polypeptides are derived from higher molecular weight precursors by a proteolytic cleavage; and secondly, this post-translational maturation is required for the building up of the crystalline structure of the trichocyst contents.

Immunological identification and characterization of chromogranins coded by poly(A) mRNA from bovine adrenal medulla and pituitary gland and human phaeochromocytoma.

Polyadenylated messenger RNA has been isolated from bovine adrenal medulla and anterior and neurointermediate pituitary gland as well as from human phaeochromocytoma. The mRNAs were translated in the presence of protease inhibitors in the rabbit reticulocyte cell-free system, containing [3H]leucine. The primary translation products were analyzed for chromogranin-like polypeptides by immunoprecipitation with chromogranin A antiserum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results show that each of the four mRNAs directed synthesis of a series of chromogranin-like proteins varying in apparent Mr between 25,000 and 120,000. The immunoreactive chromogranins coded by the three bovine mRNAs were of identical apparent Mr while the human chromogranins tended to be slightly larger. A chromogranin A-like polypeptide with apparent Mr = 72,000 was the main immunoprecipitable translation product of adrenal medulla mRNA, while that of anterior pituitary mRNA had a Mr = 87,000, that of neurointermediate pituitary mRNA a Mr = 30,000, and that of human tumor mRNA a Mr = 115,000. The results suggest that the differently sized chromogranins were coded by separate mRNAs and did not derive from a high molecular weight precursor by either nonspecific or specific post-translational protolysis.

The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor.

A 195,000 mol wt Plasmodium falciparum protein and processing fragments derived from it have been purified by monoclonal antibody affinity chromatography. A polyvalent antiserum has been raised against the purified protein and used to identify the terminal processing products associated with the merozoite. Three unique fragments of 83,000, 42,000, and 19,000 mol wt are present and they represent the major surface antigens of P. falciparum merozoites.

Discriminatory processing of the precursor forms of cytochrome P-450scc and adrenodoxin by adrenocortical and heart mitochondria.

The mitochondrial proteins involved in adrenocortical steroidogenesis are synthesized as higher molecular weight precursors which require processing by the mitochondria to their mature sizes. The post-translational maturation of two of these proteins has been examined: the cholesterol side chain cleavage cytochrome P-450 (P-450scc) and the iron-sulfur protein, adrenodoxin. Total translation products synthesized in a cell-free system programmed by bovine adrenocortical poly(A+) RNA were incubated with isolated bovine adrenocortical or heart mitochondria followed by immunoisolation of radiolabeled P-450scc or adrenodoxin. In the presence of adrenocortical mitochondria, the precursor form of P-450scc was converted into a trypsin-resistant form that had the same molecular weight as mature P-450scc. Unlike adrenocortical mitochondria, heart mitochondria were unable to process the P-450scc precursor which remained unaltered and trypsin-sensitive. In addition, a matrix fraction of heart mitochondria did not cleave the P-450scc precursor. In contrast, the adrenodoxin precursor did not exhibit similar specificity as it was processed to the mature form by both adrenocortical and heart mitochondria. Also, the adrenocortical mitochondria were not restricted to processing endogenous proteins as they imported and cleaved the precursor to ornithine transcarbamylase. The results indicate that some mitochondrial precursor proteins have tertiary structures which allow them to be recognized by all mitochondria while other mitochondrial precursor proteins have structures recognizable by only specialized mitochondria.

Uptake and Processing of in vitro Synthesized Mitochondrial Malate Dehydrogenase by Isolated Watermelon Mitochondria

Summary Mitochondrial MDH (EC 1.1.1.37) is synthesized in watermelon cotyledons as a cytosolic, higher molecular weight precursor (41 kd). We now show that the precursor produced in a reticulocyte lysate in the presence of watermelon mRNA, is posttranslationally sequestered by a crude mitochondrial fraction or by mitochondria purified on Percoll® gradients. It is processed to a proteinase resistant form corresponding to the mature subunit (38 kd). Optimal incorporations are obtained with organelles from early stages of seedling development. An uptake of the mature MDH dimer to a proteolytically protected form could not be detected.

Biosynthesis of the multiple forms of rabbit cardiac cathepsin D.

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.

Atriopeptins: A family of potent biologically active peptides derived from mammalian atria

Extracts of rat atria are potent stimulators of sodium and urine excretion, and relax vascular and intestinal smooth muscle preparations. The structures of six biologically active peptides obtained from atrial extracts are reported here. Ion exchange chromatography of a low molecular weight fraction obtained by gel filtration of atrial extracts produced two natriuretic fractions: the first induced relaxation of intestinal smooth muscle strips only, whereas the second also relaxed vascular strips as well. From the first fraction four pure biologically active peptides obtained by reverse phase HPLC have been sequenced: the 21 amino acid peptide, designated atriopeptin I, and three homologs (des-ser 1-, des-ser 1-ser 2-, and des-ser 21-atriopeptin I). From the second fraction two pure biologically active peptides were obtained, which had C-terminal extensions of atriopeptin I: atriopeptins II (23 amino acid residues) and III (24 residues), having respectively phe-arg and phe-arg-tyr C-termini. These results suggest that this family of six peptides, sharing the same 17 membered ring formed by an internal cystine disulfide, is derived from a common high molecular weight precursor.

Synthesis of the Crystalloid Protein Complex In Vivo in the Endosperm of Developing Castor Bean Seeds

The synthesis of subunit polypeptides of the crystalloid protein complex has been examined in endosperm from developing castor bean (Ricinus communis L. cv Hale) seeds. Pulse-label and -chase studies in vivo have shown that synthesis initially involves the formation of high molecular weight precursors (50 to 60 kilodaltons) comprising peptide-linked acidic and basic polypeptides. Precursor processing involves the posttranslational cleavage of the peptide bond to yield authentic and polypeptides. This processing has a half-life of 35 to 40 minutes and is preceded by a 45- to 60-minute lag period. Both precursor and subunit polypeptides are shown to exhibit similar molecular weight and pI heterogeneity, and this is suggested to be due to the expression of a multigene family.