Pneumocystis carinii f. sp. carinii synthesizes de novo four homologs of ubiquinone

Ubiquinone, coenzyme Q, plays a pivotal role in electron transport and is a target for chemotherapy against a number of eukaryotic infectious agents, including Pneumocystis carinii. Coenzyme Q10 was previously identified as the major ubiquinone homolog in P. carinii isolated and purified from rat lungs; CoQ9 was also present. In contrast, CoQ9 and CoQ8 (but not CoQ10) were detected in the lungs of uninfected rat controls. These observations suggested that the pathogen synthesizes CoQ10, and perhaps CoQ9 as well. In the present study, CoQ biosynthesis in P. carinii was examined in greater detail. Radiolabeled mevalonate, a precursor of the CoQ polyprenyl chain, was incorporated in vitro into P. carinii ubiquinones. Incorporation of radiolabeled mevalonate into P. carinii CoQ was not enhanced by treating cells with lovastatin, suggesting that the cells did not transport the drug, or that a lovastatin-insensitive pathway for de novo synthesis of isoprenoids may also function in this organism. Radiolabeled precursors of the ring moiety, including shikimic acid, p-hydroxybenzoic acid, and tyrosine were also incorporated into P. carinii CoQ. Unexpectedly, it was found that not only CoQ9 and CoQ10, but also CoQ7, and CoQ8, were metabolically radiolabeled by all the precursors tested, indicating that the organism synthesizes CoQ7, CoQ8, CoQ9, and CoQ10. Metabolic radiolabeling of ubiquinones in rat lung controls was not detected in experiments using either radioactive mevalonate or p-hydroxybenzoate. Thus the incorporations measured using purified P. carinii preparations were due to the enzymes of the organism.     

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.     

Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.     

Metabolism of coenzyme Q10: biliary and urinary excretion study in guinea pigs.

Biliary and urinary metabolites were examined after intravenous administration of 14C-coenzyme Q10 (14C-CoQ) to guinea pigs. Cumulative recovery of administered radioactivity for up to 8 hours by bile drainage was 4.8%. The greater part of radioactivity was detected in conjugate form. After hydrolyzing with beta-glucuronidase, aglycone fragments were subjected to methylation and reductive acetylation. The main metabolite was demonstrated to be Q acid-1 1,4-hydroquinone diacetate methyl ester (M-1) on HPLC. Then, the main metabolite was assumed to be glucuronide of 2,3-dimethoxy-5-methyl-6-(3'-methyl-5'-carboxy-2'-pentenyl)-1, 4-benzohydroquinone [Q acid-I hydroquinone]. The cumulative urinary recovery of the administered radioactivity over 48 hours was 8.3%. The labeled samples were treated similarly to bile. The urinary metabolites of CoQ10 consisted of unconjugated and conjugated forms. Lyophilized urine was treated as a bile sample and analyzed. The two major metabolites were assigned to be M-1 and Q acid-II 1,4-hydroquinone diacetate methyl ester (M-2). Then, the two metabolites were assumed to be composed of Q acid-I and 2,3-dimethoxy-5-methyl-6-(3'-carboxypropyl)-1,4-benzoquinone (Q acid-II) in free and corresponding hydroquinone conjugate forms. To investigate the effect of exogenous labeled CoQ10 on unlabeled CoQ10 (endogenous) metabolites in urine, simultaneous quantitative determination was performed using deuterium labeled CoQ10 (CoQ10-d5). Urine collected over a 72-hour period after intravenous administration of CoQ10-d5 was processed similarly to that described above and two derivatized metabolites (M-1 and M-2) were quantified by gas chromatography-mass fragmentography with the multi-ion detection method. The analytical results showed that the addition of exogenous labeled CoQ10 did not influence the metabolism (or breakdown) of unlabeled (endogenous) CoQ10.     

Preparation, characterization, and microbial degradation of specifically radiolabeled [C]lignocelluloses from marine and freshwater macrophytes

Specifically radiolabeled [C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [C]phenylalanine, [C]tyrosine, and [C]cinnamic acid as precursors. Specifically radiolabeled [C-polysaccharide]lignocelluloses were prepared by using [C]glucose as precursor. The rates of microbial degradation varied among [C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [C]phenylalanine and [C]tyrosine were found associated with protein, although very little (3%) radioactivity from [C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to CO(2); during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

核酸探针中放射性同位素的快速检测

利用尼龙膜作为介质,以0．4mol／L的NaOH为层析液,进行膜上层析,分离经放射性同位素标记的核酸探针和未掺入的放射性游离单核苷酸,再经放射性强度测定即可计算出标记后的核酸探针的放射性比活。方法简单快捷,产生的放射性废物少,完全可以替代经典的三氯乙酸沉淀法及滤膜吸附法。     

Ubiquinone synthesis in mitochondrial and microsomal subcellular fractions of Pneumocystis spp.: differential sensitivities to atovaquone

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ7, CoQ8, CoQ9, and CoQ10, as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.     

Time course of distribution to tissues of 125I-somatomedin A injected into the rat

125I-somatomedin A (SMA) was injected iv into rats. Distribution studies in rats showed concentrations of radioactivity to be high in kidney and plasma, low in brain, and intermediate in other tissues. The concentration of total and trichloracetic acid (TCA) precipitable radioactivity in rat blood and tissues fell at rapid rate. Ninety per cent of the radioactivity was in the urine in 24 hr, and only 15% of urine radioactivity was TCA precipitable. The half-life of the radioactivity in TCA-precipitable fraction from blood and that from tissues were nearly identical (about 6 hr). In both liver and kidney, TCA-precipitable radioactivity was detected in membrane and/or organellar fraction and cytosol fraction. Sephadex G-200 chromatography at neutral PHY AT NEUTRAL PH of plasma after injection of 125I-SMA revealed 3 peaks of radioactivity in higher molecular weight region than purified SMA.     

Separation and determination of liver uric acid and allantoin

We previously described the only satisfactory procedure yet achieved for separating uric acid and allantoin from rat liver. The procedure was based on trichloroacetic acid (TCA) extraction, acid hydrolysis, treatment with Hg-acetate, and cation- and anion-exchange chromatography. After separation, allantoin was quantified by a colorimetric method, and uric acid enzymatically using uricase. Since this procedure is too time-consuming, we propose an improved version which avoids the need for anion-exchange chromatography and the complex assay of catabolic compounds. The new method consists of a very fast and simple HPLC separation and direct determination of uric acid and allantoin at 220 nm. The method can be used for fresh tissue or after treatment of the tissue with labeled precursor.     

Application of radiolabeled tracers to biocatalytic flux analysis.

Radiolabeled tracers can provide valuable information about the structure of and flux distributions in biocatalytic reaction networks. This method derives from prior studies of glucose metabolism in mammalian systems and is implemented by pulsing a culture with a radiolabeled metabolite that can be transported into the cells and subsequently measuring the radioactivity of all network metabolites following separation by liquid chromatography. Intracellular fluxes can be directly determined from the transient radioactivity count data by tracking the depletion of the radiolabeled metabolite and/or the accompanying accumulation of any products formed. This technique differs from previous methods in that it is applied within a systems approach to the problem of flux determination. It has been used for the investigation of the indene bioconversion network expressed in Rhodococcus sp. KY1. Flux estimates obtained by radioactive tracers were confirmed by macroscopic metabolite balancing and showed that indene oxidation in steady state chemostat cultures proceeds primarily through a monooxygenase activity forming (1S,2R)-indan oxide, with no dehydrogenation of trans-(1R,2R)-indandiol. These results confirmed the significance of indan oxide formation and identified the hydrolysis of indan oxide as a key step in maximizing the production of (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixivan.     

De novo biosynthesis of polyunsaturated fatty acids in the cockroach Periplaneta americana

The de novo biosynthesis of 6,9,12-linolenic acid, 11,14-eicosadienoic acid, 5,11,14-eicosatrienoic acid, and arachidonic acid was demonstrated in adult female cockroaches, Periplaneta americana. These four polyunsaturated fatty acids (PUFA) were present primarily in the phospholipid (PL) fraction of both males and females. They were purified by AgNO3 thin-layer chromatography and high pressure liquid chromatography. The double bond positions of the major isomer of eicosatrienoic acid were shown to be at the delta 5,11,14 positions by gas chromatography-mass spectrometry (GC-MS) of both methoxy and epoxide derivatives and gas-liquid chromatography (GLC) and GC-MS of ozonolysis products. The other PUFAs cochromatographed with standards on both packed and capillary GLC columns. The in vivo incorporation of [1-14C]acetate into 5,11,14-eicosatrienoic acid, 11,14-eicosadienoic acid, 6,9,12-linolenic acid, and arachidonic acid was demonstrated by radio-GLC and radio-HPLC and for 5,11,14-eicosatrienoic acid by radio-GLC of ozonolysis products. The latter technique clearly demonstrated that the entire eicosatrienoic acid molecule was labeled. Thoracic tissue contained the highest amount of radiolabeled 5,11,14-eicosatrienoic acid (1.6% of total radioactivity incorporated into PL) while radiolabeled 11,14-eicosadienoic acid was found primarily in abdominal epidermal tissue (2% of total radioactivity incorporated into PL). Radiolabeled arachidonic and 6,9,12-linolenic acids comprised 0.1 and 0.02%, respectively, of the total radioactivity in the PL fraction. These data document the de novo biosynthesis of di-, tri-, and tetraunsaturated fatty acids in the American cockroach, and indicate that this animal can desaturate on both sides of the delta 9 double bond of oleic acid.     

Evidence for non-equilibrating pools of apolipoprotein C-III in plasma lipoproteins

Using immunoaffinity chromatography to isolate apoC-III from radiolabeled lipoproteins for direct determination of specific radioactivity, we have studied the metabolism of human apoC-III in VLDL and in HDL following the bolus injection of 125I-labeled VLDL. Transfer of apoC-III radioactivity from VLDL to HDL was detected in the plasma sample drawn 5 min after injection of the tracer. However, the specific radioactivity of apoC-III in VLDL was found to be higher than that in HDL, with this difference being maintained throughout the sampling period (48-72 hr). The ratios of the respective specific activities ranged from 1.2 to 1.9 in six subjects studied (two normolipidemics and four hypertriglyceridemics). When 125I-labeled HDL was injected as the tracer, however, the higher apoC-III specific radioactivity was associated with the HDL fraction. This lack of complete equilibration of apoC-III between VLDL and HDL in vivo was further characterized by in vitro studies using either 125I-labeled VLDL or 125I-labeled HDL. All incubations were carried out for 3 hr at 37 degrees C followed by 16 hr at 4 degrees C and the apoC-III specific activity in each lipoprotein fraction was directly determined after immunoaffinity chromatography. In a study of plasma from a mildly hypertriglyceridemic subject in which 125I-labeled VLDL was incubated with unlabeled HDL, apoC-III specific activities in VLDL remained 30% greater than that in HDL. When 125I-labeled HDL (from the same subject) was incubated with unlabeled VLDL of apoC-III, final specific activity in VLDL was less than 10% of that of HDL apoC-III. Differences in specific activities were also demonstrated when radiolabeled purified apoC-III was exchanged onto VLDL prior to its incubation with HDL. A consistent difference in apoC-III specific activities in VLDL and HDL was observed after isolation of the particles either by molecular sieve chromatography or by ultracentrifugation. These studies demonstrated that, while the exchange of apoC-III between VLDL and HDL may be very rapid, this equilibration is not complete. Pools of apoC-III that do not participate in the equilibration process are present in both the VLDL and HDL fractions and could account for 30-60% of the total apoC-III mass in each lipoprotein fraction.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

Studies on the bioactivity of radiolabeled,highly-purified bovine thyrotropin

Bovine thyrotropin radiolabeled stoichiometrically with chloramine T was subjected to high pressure liquid chromatography on a Waters' Protein I-125 column. More than 80% of the radioactivity eluted after the Na¹²⁵I (“salt”) peak. In contrast, thyrotropin bioactivity eluted before the salt peak. Radiolabeled thyrotropin affinity-purified with thyroid plasma membranes eluted after the salt peak. Discordance between the thyrotropin bioactivity and radioactivity elution profiles was confirmed by labeling thyrotropin with ¹²⁵I by lactoperoxidase and then measuring both bioactivity and radioactivity in each chromatographic fraction. These data suggest that the bioactivity in radiolabeled thyrotropin may not be inherent in the radiolabeled molecules.     

Plasma clearance and tissue distribution of radiolabeled leptin in the chicken

Leptin is an adipose and liver tissue-derived secreted protein in chickens that has been implicated in the regulation of food intake and whole-body energy balance. In this study, the metabolic clearance and tissue uptake of leptin were examined in the chicken (Gallus gallus). Four-week-old broiler males were infused with (125)I-labeled mouse leptin. Chromatography of radiolabeled leptin in plasma produced two peaks, one at 16 kDa (free leptin) and a free iodine peak. No leptin binding protein in blood was detected. Leptin was cleared with a half-life estimate of 23 min. In order to investigate the tissue distribution and uptake of radiolabeled leptin, multiple tissues were removed from infused birds at 15 and 240 min post-infusion, and trichloroacetic acid (TCA)-precipitable radioactivity was determined. The amounts of radioactivity at 15 min post-infusion in the tissues in rank order were: kidney, testis, lung, spleen, heart, liver, small and large intestine, gizzard, pancreas, bursa, leg and breast muscle, adrenals, and brain. A slightly different pattern of distribution was observed at 240 min post-infusion. We conclude from these studies that unlike mammals, no circulating leptin binding protein is present in chickens. Leptin is metabolized and cleared very rapidly from blood by the kidney.     

Paper disc estimation of radioactive RNA: studies on the presence and elimination of metabolically generated artifacts from labeled purine and pyrimidine precursors

Conventional usage of the filter-paper disc technique following in vivo hepatic incorporation of certain labeled pyrimidine and purine RNA precursors resulted in the presence of radioactivity on the filter disc in species other than RNA. Thus the filter paper disc technique, conventionally conducted, may be misleading. To eliminate artifacts, it is proposed that replicate filter discs be processed, one group passing through the usual cold TCA washings only, while a parallel group of replicate discs are heated for 20 min at 90°C in 5% TCA to remove RNA. True RNA radioactivity is the difference of counts from the cold TCA precipitable material and the radioactivity remaining after hot TCA extraction.     

Simplified assay for lysyl oxidase activity.

A simplified method for the assay of lysyl oxidase activity was developed. The method is based on the measurement of tritiated water released by enzyme action from labeled protein-bound lysine and hydroxylysine. Trichloroacetic acid (TCA) supernates of the incubation mixtures are passed through small Dowex 50 (H⁺) columns and the effluents are counted. For rapid screening purposes an indication of the presence of enzyme activity in enzyme preparations can be obtained by measuring the radioactivity present in aliquots of the TCA supernates as such and by measuring the radioactivity after drying at 60°C, taking the difference between the two as a measurement of enzyme activity.     

Utilization of the heme moiety of hemoglobin by Schistosoma mansoni schistosomules in vitro

The hemoglobin in mouse reticulocytes was labeled in vitro with either [3H], [14C] aminolevulinic acid (ALA), or [3H] leucine. Specific labeling of the globin moiety with labeled leucine, and the heme moiety with labeled ALA, was confirmed by carboxymethylcellulose chromatography and cyclohexanone extraction. Most of the leucine label recovered from reticulocytes that were incubated for 4 hr was incorporated in hemoglobin. However, 2 hr incubation of reticulocytes in the presence of labeled ALA followed by 4 hr in cell incubation medium in the absence of ALA was required for sufficient incorporation of the radionuclide into reticulocyte hemoglobin. In all reticulocyte labeling experiments, regardless of the radionuclide used, label was also observed in non-hemoglobin heme-containing molecules. Schistosoma mansoni schistosomules fed reticulocytes in vitro in which the heme moiety of hemoglobin was labeled displayed radioactivity in the protein fraction of the organisms, as determined by TCA precipitation, and in the ethanol-soluble component. In comparison, schistosomules fed reticulocytes containing globin-labeled hemoglobin displayed radioactivity only in the protein component. Pre-incubation of the schistosomules in puromycin prior to exposure to lyophilized, [14C] ALA-labeled hemoglobin partially inhibited incorporation of label. These results suggest that the organism utilizes not only the globin moiety of hemoglobin in its nutritional requirements, but the heme moiety as well.     

Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered, indicating that bacitracin acted predominantly at the cell surface. In the presence of chloroquine, which dramatically increased the contribution of peak I to specific binding, 'intact' insulin was substantially overestimated when determined as the TCA-precipitable fraction. In addition, all peak I material and 50% of cell-associated label in peak II was trapped intracellularly, thereby pointing to the lysosomal or prelysosomal site of action of this drug.     

Myeloperoxidase precursors incorporate heme

Myeloperoxidase of neutrophil granulocytes is synthesized as a larger molecular weight precursor, which is processed to yield mature polypeptides with molecular weights of 62,000 and 12,000. We have investigated the incorporation of heme into myeloperoxidase of the human promyelocytic HL-60 cell line labeled with 5-amino[14C]levulinic acid. Myeloperoxidase was isolated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radiolabeled myeloperoxidase was visualized by fluorography. A 3-h pulse labeling with 5-amino[14C]levulinic acid resulted in labeling of the Mr 90,000 and Mr 82,000 precursor polypeptides. During subsequent chase of the label, conversion to mature radioactive heavy Mr 62,000 subunit was observed but no radioactivity was associated with the mature small Mr 12,000 subunit. Peptide mapping after proteolytic cleavage with V8 proteinase showed that 5-amino[14C]levulinic acid was associated with a single Mr 23,000 polypeptide while multiple radioactive fragments were visible after proteolytic cleavage of myeloperoxidase biosynthetically labeled with [14C]leucine. That 5-amino[14C]levulinic acid was specifically incorporated into heme of myeloperoxidase was also demonstrated by dissociation under reducing conditions which yielded 14C-labeled heme as indicated by reversed phase high pressure liquid chromatography. The ionophore monensin and the base chloroquine, which block processing of myeloperoxidase, did not affect the incorporation of 5-amino[14C]levulinic acid, further supporting the notion that the incorporation of heme is independent of final processing of the polypeptide. Our data establish that heme is incorporated into myeloperoxidase already at the level of the precursor and that processing yields a heme-containing heavy subunit and a heme-free small subunit.