GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms

The chaperonin GroEL is a ribosome-sized double-ring structure that assists in folding a diverse set of polypeptides. We have examined the fate of a polypeptide during a chaperonin-mediated folding reaction. Strikingly, we find that, upon addition of ATP and the cochaperonin GroES, polypeptide is released rapidly from GroEL in a predominantly nonnative conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound rapidly by other GroEL molecules. Folding appears to occur in an all-or-none manner, as proteolysis and tryptophan fluorescence indicate that after rebinding, polypeptide has the same structure as in the original complex. These observations suggest that GroEL functions by carrying out multiple rounds of binding aggregation-prone or kinetically trapped intermediates, maintaining them in an unfolded state, and releasing them to attempt to fold in solution.     

Characterization of pathotype-specific epitopes of newcastle disease virus fusion glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and post-source decay sequencing

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) was used to characterize the F2 polypeptide of the fusion (F) protein of an avirulent isolate (VRI 82-6409) of Newcastle disease virus (NDV) that was previously identified by immunochemical screening as having a variant cleavage activation sequence in its fusion protein precursor (F0). The major glycoform of the intact F2 polypeptide of the VRI 82-6409 isolate was 89 Da smaller than the F2 polypeptide of the avirulent V4 isolate of the Queensland strain of NDV. Analysis of AspN protease digests of the F2 polypeptides by MALDI/TOF-MS, with and without high-performance liquid chromatographic (HPLC) separation, showed this mass difference to be due to a combination of differences in the extents of glycosylation and an amino acid difference in the AspN peptides derived from the C-termini of the F2 polypeptides. Accuracies achieved in analysis of the AspN peptides allowed the identification of this amino acid difference as glutamic acid in the VRI 82-6409 isolate compared with glycine in the V4 isolate. Analysis of fragments formed by post-source decay (PSD) of ions of the C-terminal AspN peptides localized the difference to the C-terminal residues of the respective F2 polypeptides. The present study demonstrated that MALDI/TOF-MS is a highly effective technique for the characterization of NDV variants identified by immunochemical screening of pathotype-specific epitopes at the C-termini of their F2 polypeptides.     

Short-term effects of naltrexone in 155 heroin ex-addicts

The narcotic antagonist naltrexone was administered for periods of up to 8 months to a total of 155 patients at a dose of 40-200 mg per day. The antagonistic effect of naltrexone was tested by injections of heroin. Eighty milligrams of natrexone was effective for 48hr. The antagonistic effect decreased at 72 hr after the administration of 120-200 mg of naltrexone. Laboratory tests indicated no signs of toxicity. Naltrexone may elicit an increase in blood pressure and opigastric pain. Neither of these side effects appear clinically important. No signs of dependence on naltrexone were detected. These results suggest that naltrexone may be useful for clinical treatment of opiate dependence.     

Intermediates in adenovirus assembly

Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII.     

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.     

Naltrexone: disposition, metabolism, and effects after acute and chronic dosing

The disposition of naltrexone during acute and chronic administration of 100-mg oral dose was studied in 4 subjects. Following an acute dose the mean (X) peak naltrexone plasma level was 43.6 +/- 29.9 ng/ml at 1 hr and for the major biotransformation product, beta-naltrexol, was 87.2 +/- 25.0 ng/ml at 2 hr. Twenty-four hours after the dose the X levels of naltrexone and beta-naltrexol declined to 2.1 +/- 0.47 and 17.6 +/- 5.0 ng/ml, respectively. Following chronic administration and X peak plasma levels of naltrexone and beta-naltrexol rose to 46.4 +/- 18.5 and 158.4 +/- 89.9 ng/ml at 1 hr, but by 24 hr both compounds declined to levels of the same order as in the acute state at 24 hr. Plasma levels of naltrexone and beta-naltrexol measured 24 hr after the daily doses of naltrexone throughout the study indicated that steady-state equilibrium was rapidly attained and that there was no accumulation of naltrexone and beta naltrexol in the plasma after chronic treatment on 100 mg oral doses. Biexponential kinetics were observed for naltrexone and beta-naltrexol in the first 24 hr. The half-life of naltrexone and beta-naltrexol decreased slightly from the acute to thechronic study from 10.3 +/- 3.3 to 9.7 +/- 1.1 hr and from 12.7 +/- 2.6 to 11.4 +/- 2.0 hr. The plasma levels of naltrexone declined slowly from 24 through 72 hr from 2.4 to 1.7 ng/ml, with an apparent half-life of 96 hr. The renal clearance data indicate that naltrexone is partially reabsorbed while beta naltrexol is actively secreted by the kidney. During acute and chronic naltrexone administration the mean fecal excretion was 2.1% and 3.6% while urinary excretion was 38% and 70% of the dose in a 24-hr period. Opiate antagonism to 25 mg heroin challenges was nearly complete through 48 hr after naltrexone. At 72 hr the objective responses reappeared to a greater extent than the subjective ones. Correlation coefficient (r) between naltrexone plasma levels and opiate antagonism was 0.91 and between individual half-life of naltrexone and opiate antagonism it was 0.99.     

Development of Chronomers tm for narcotic antagonists

The object of this program is to prepare a bioerodable naltrexone delivery system which can be implanted subcutaneously in humans and which can relieve the narcotic antagonist over 1-6 months at relatively constant and sufficient rates to block the euphoric effect of morphine based drugs. The system is composed of naltrexone uniformly dispered in a solid hydropholic CHRONOMER TM matrix which undergoes predictable surface erosion when exposed to an aqueous medium. Kinetic studies in vitro have been carried out during the course of the program to determine the best composition for the system. Toxilogical studies conducted at ALZA during the past 2 years have not revealed limiting adverse effects of either the CHRONOMER TM materials or their hydrolysis products. The tail-flick test procedure was used to measure the effectiveness of naltrexone to antagonize the analgesis of morphine in rats. Naltrexone infused intravenously at doses of 4 and 16 ug/kg/hr resulted in, after 6 hours, 54 and 89% antagonism, respectively, against a 63.5% effective dose of morphine. Perliminary sterilization studies showed that no adverse effects to CHRONOMER TM/naltrexone systems occurred after exposure to 2.5 or 5.0 mrads of 60CO irradiation.     

Mammalian cytosolic DnaJ homologues affect the hsp70 chaperone-substrate reaction cycle, but do not interact directly with nascent or newly synthesized proteins

Members of the hsp70 family of molecular chaperones interact with and stabilize nascent polypeptides during synthesis and/or translocation into organelles. The bacterial hsp70 homologue DnaK requires the DnaJ cofactor for its reaction cycle with polypeptide substrates. DnaJ stimulates the ATPase activity of the DnaK chaperone and thereby is thought to regulate the affinity of DnaK for its protein target. Herein we have analyzed some of the biochemical properties of two mammalian cytosolic DnaJ homologues, the hdj-1 and hdj-2 proteins. We were particularly interested in examining the proposal that DnaJ homologues are the first molecular chaperones to interact directly with nascent polypeptides. Nascent/newly synthesized proteins, nascent polypeptides released from the ribosome by puromycin, or polypeptides misfolded as a result of incorporation of an amino acid analogue were not found in complexes with either of the two HeLa cell DnaJ homologues. We still were unable to demonstrate any interactions between hdj-1p and nascent/newly synthesized proteins even after chemical cross-linking. We did find that hdj-1p, like bacterial DnaJ, stimulated the ATPase activity of hsp70. Stable complex formation between hsp70 and an unfolded polypeptide substrate in vitro was found to be reduced in the presence of hdj-1p and ATP. Thus, while hdj-1p likely does function as a cofactor for the hsp70 chaperone, having effects on hsp70's ATPase activity and conformation/oligomeric structure and the stability of hsp70-substrate complexes, it was not observed to interact directly with nascent/newly synthesized proteins. Rather, hdj-1p likely serves a regulatory role, governing the reaction cycle of hsp70 with polypeptide substrates.     

Bunyamwera virus-induced polypeptide synthesis

Bunyamwera virus-induced polypeptide synthesis in BSC-1 cell has been studied using polyacrylamide gel electrophoresis and autoradiography. Four virus-induced polypeptides were identified. Their molecular weights were 200 X 10(6) (L), 128 X 10(6) (G1), 31 X 10(6) (G2), and 23 X 10(6) (N). Pulse-chase experiments, short labeling experiments, and experiments using amino acid analogs failed to show evidence of polypeptides processing by proteolytic cleavage. Analysis of the kinetics of synthesis of these polypeptides showed that a clear division into early and late categories could be made, the onset of synthesis of polypeptide N and L rapidly reached a peak and then declined. Polypeptides G1 and G2 were made for several hours; their rate of synthesis then declined. All four polypeptides then continued to be made in relatively small amounts for many hours.     

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. I. Determination of average rates of polypeptide chain elongation

Average rates of polypeptide chain elongation have been determined in cockerel liver explants on 4 successive days following an in vivo injection of 17 beta-estradiol. Incorporation of [3H]leucine by the explants is linear for at least 24 h, and the rate of protein synthesis increases significantly after estrogen injection. The explants synthesize and secrete serum albumin. B-apolipoprotein, and the phosphoprotein vitellogenin at relative rates which are similar to those reported for liver in vivo. Using this system, changes in the average rates of polypeptide chain elongation have been analyzed as a temporal sequence following a single injection of 17 beta-estradiol into cockerels. For this, average ribosome half-transit times were determined by measuring the kinetics of transfer of labeled polypeptides from polysomal-bound to released polypeptides. The data revealed a dramatic effect of estradiol on the average ribosome half-transit time with a maximum increase of 4.6-fold; however, the average size of polypeptides synthesized by explants at the peak of induction increased only 15% when compared to uninduced liver explants. These findings indicate that injection of estradiol results in large changes in the actual rates at which amino acids are added to the growing nascent polypeptide chain; that is, rates of polypeptide chain elongation. Therefore, translation-level regulation of protein synthesis in cockerel livers plays a significant part in determining the magnitude of the response to hormone stimulation.     

Formation of high axial ratio microstructures from peptides modified with glutamic acid dialkyl amides

A growing number of amphiphiles are known to form high axial ratio microstructures (HARMs) such as the hollow cylindrical microstructures called lipid tubules. As a prelude to exploring the potential of HARMs formed from lipopeptides in controlled release drug delivery, several microstructure formation conditions were investigated. We report the preparation of several glutamic acid dialkyl amides with varying alkyl chain lengths bearing a verity of peptides (1-4 amino acids) [peptide-Glu-(NHCnH2n+1)2, n=12, 14, 16]. These surfactants have been rapidly and efficiently converted into HARMs in aqueous buffer at physiological pH and ionic strength, or in buffer containing MeOH or EtOH. Helical ribbons and tubular HARMs were produced that were stable for as long as 6 months below the phase transition temperatures of the compounds. To estimate the stability of HARMs in vivo, HARMs formed from (Pro)3-Glu(NHC16H33)2 were incubated with DOPC liposomes or fetal calf serum at 40 degreesC. HARM size and shape did not change significantly, suggesting that such lipopeptide particles can retain their morphology long enough in vivo to be useful as drug delivery vehicles.     

Chronic l-alpha acetylmethadol in rhesus monkeys: discriminative stimulus and other behavioral measures of dependence and withdrawal

This study characterized discriminative stimulus and other effects of naltrexone in rhesus monkeys treated daily with the long-acting opioid l-alpha acetylmethadol (LAAM). An initial dose-finding study assessed the rate-decreasing effects of naltrexone in three monkeys receiving LAAM daily (0.32-1.78 mg/kg); subsequently, these monkeys and a fourth received 1.0 mg/kg/12 hr of LAAM although discriminating between naltrexone and saline. Responding occurred on the saline lever after the administration of LAAM, whereas >90% drug-lever responding occurred after the administration of 0.1 mg/kg of naltrexone that also elicited signs of withdrawal. Naloxone and quadazocine, but not morphine, nalbuphine or ketamine, substituted for naltrexone. Morphine and nalbuphine shifted the naltrexone dose-effect curve to the right. Compared to precipitated withdrawal, deprivation-induced withdrawal occasioned less naltrexone-lever responding and fewer observable signs of withdrawal. Maximal naltrexone-level responding occurred 24 to 48 hr after the discontinuation of LAAM treatment; the frequency of other withdrawal signs also peaked 24 to 48 hr after the discontinuation of LAAM. Partial naltrexone-lever responding occurred for up to 10 days after discontinuation of LAAM treatment; 4 and 8 days after the discontinuation of LAAM treatment, 0.1 mg/kg of naltrexone did no further increase naltrexone-lever responding or withdrawal signs suggesting that less-then-maximal naltrexone-lever responding was not due to long-lasting effects of LAAM or its metabolites. The discriminative stimuli that are associated with LAAM deprivation might be different from the stimuli associated with either training condition. This study is the first antagonist discrimination in non-humans primates treated chronically with LAAM and the results indicate that the naltrexone stimulus is related to opioid withdrawal.     

Isolation and characterization of a 20 kD prolamin from kodo millet (Paspalum scrobiculatum) (L.): homology with other millets and cereals

The homologus 20 kD prolamin from kodo millet and other minor millets viz. barnyard, little and foxtail millets, were purified using preparative gel electrophoresis and reversed phase high performance liquid chromatography (RP-HPLC). The amino acid composition of the purified 20 kD prolamin protein from different minor millets revealed higher content of glutamic acid, alanine, leucine and serine and lower quantity of lysine and methionine. They contain 55 to 58 percent of non-polar amino acids which make them more hydrophobic than other protein fractions. The total number of amino acid residues per polypeptide chain ranged from 152 to 155 based on theoretical calculation. Peptide mapping of the 20 kD prolamin hydrolyzed with trypsin gave fewer cleavage products than expected. The antigenic relationships among these minor millets and cereals viz. wheat, maize, rice, sorghum, finger millet and pearl millet were studied using the antibody raised against the 20 kD prolamin. Cross reactivity was seen in all the minor millets at the 20 kD region. But in barnyard and little millets lower molecular weight polypeptides also cross reacted with the antibody. Immunoblotting studies revealed that the prolamins from other cereals and millets are related to the 20 kD prolamin of kodo millet. Rice was the only common cereal that did not cross react immunologically with the antibody raised against 20 kD prolamin of kodo millet.     

Translation in a reticulocyte cell-free system of RNA isolated from blood and culture forms of Trypanosoma brucei

RNA with messenger activity has been extracted from both blood and culture (insect mid-gut) forms of Trypanosoma brucei and translated in a reticulocyte cell-free system. The products of this cell-free system have been compared, and many common polypeptides demonstrated. A major polypeptide of 58000--65000 molecular weight was made when both blood and culture form RNA was added to the cell-free system. Antiserum raised against purified variant antigen from a cloned variant (MIAG 099) was used to detect specific products of this system. A major polypeptide of approximately 58000-65000 molecular weight was precipitated when the homologous trypanosome (MIAG 099) blood form RNA was used in the cell-free system. No such polypeptide was precipitated when RNA from a heterologous strain culture or blood form was used in the system. Competition experiments, in which excess purified variant antigen was addded after incubation but before addition of specific antiserum, confirmed that the polypeptide of 58000--65000 molecular weight is the variant antigen.     

The opioid receptor antagonist nalmefene reduces responding maintained by ethanol presentation: preclinical studies in ethanol-preferring and outbred Wistar rats

Nalmefene, the 6-methylene derivative of naltrexone, was examined after subcutaneous (s.c.) (0.0001 to 8.0 mg/kg) and oral (10 to 80.0 mg/kg) administration in ethanol (EtOH)-preferring rats whose responding (i.e., lever pressing) was maintained by the presentation of EtOH. Naltrexone (0.01 to 40 mg/kg) was used as a reference opioid antagonist. EtOH (10% v/v) and saccharin (0.025 to 0.1% w/v) solutions were concurrently available for 1 hr each day under a two-lever, fixed-ratio schedule in which four responses on one lever produced the EtOH solution and four responses on the other lever produced the saccharin solution. When basal response rates for saccharin were 10% that of EtOH, all routes of nalmefene administration reduced control levels of responding maintained by EtOH by 38 to 84%. When basal response rates for saccharin-maintained responding were 60% or 82% that of EtOH, only lower s.c. naltrexone (e.g., 0.01 to 0.025 mg/kg) and nalmefene (e.g., 0.01 to 0.10 mg/kg) doses produced a selective dose-dependent suppression of EtOH-maintained responding. Higher nalmefene (0.25 to 8.0 mg/kg) and naltrexone (1.0 to 20.0 mg/kg) doses failed to produce a dose-dependent suppression on EtOH or saccharin maintained responding. Both antagonists suppressed responding maintained by EtOH primarily during the initial 10-min period, with little additional suppression occurring across the remainder of the 60-min period. Subcutaneous nalmefene was 3200- to 6400-fold more potent than oral nalmefene, suggesting bioavailability was optimized using the s.c. route. Nalmefene (0.5 mg/kg, s.c.) treatment for 10 consecutive days produced mild tolerance development, whose effects dissipated by day 8. Naltrexone (10 to 40 mg/kg) and nalmefene (1.5 to 3.0 mg/kg), given 8 to 24 hr before the test session, reduced control levels of responding maintained by EtOH by 82%. Thus, immediate opioid receptor occupancy was not required to observe antagonism. These data demonstrate that, under a variety of experimental conditions, nalmefene is an effective antagonist of responding maintained by EtOH and lend support to clinical reports that nalmefene may function as an alternative pharmacotherapy to naltrexone to reduce EtOH-motivated behavior and prevent relapse.     

Testing of drug delivery systems for use in the treatment of narcotic addiction

The evaluation of the drug release characteristic of four naltrexone delivery systems has been carried out together with the development of analytical techniques and an investigation of the metabolic profile of naltrexone. Pharmacologic evaluation of the four delivery systems in the mouse indicated significant analgesic antagonism for a period of from 16-22 days. Further evaluation of one of these systems by measurement of the rate of excretion of radioactivity after administration of radiolabelled naltrexone in the delivery system confirmed that significant release occurs for a time period of about 15 days. Electron capture gas-liquid chromatographic assays for naltrexone and naloxone in plasma or urine have been developed that yield linear calibration curves and are sensitive to one ng/ml. Studies on naltrexone disposition indicate that (a) binding to plasma proteins in several species varies from 20-26 per cent, (b) distribution of drug from blood is extremely rapid and extensive, (c) beta-naltrexol is a major metabolite of naltrexone in man, monkey and guinea pig among six species studied, whereas alpha-naltrexol is a minor metabolite in the monkey and guinea pig only, and (d) metabolic reduction of naltrexone occurs in the 100,000 x g supernatant of guinea pig liver. Pharmacokinetic studies of naltrexone in the dog and monkey indicate that the drug is rapidly distributed and eliminated, has a very large apparent volume of distribution and a total body clearance greater than the rate of liver blood flow.     

Labeling of a major fibroblast surface protein (fibronectin) catalyzed by blood coagulation factor XIIa

Incubation of cultured human fibroblasts with blood coagulation factor XIIIa (plasma transglutaminase, fibrinoligase) and the fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, resulted in fluorescent labeling of three cellular polypeptides. The molecular weights of the labeled polypeptides, estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction, were: greater than 1.2-10(6), 2.2-10(5), and 1.3-10(5). The labeled 2.2-10(5) dalton polypeptide was susceptible to mild trypsinization and not present in cultures of SV-40 transformed fibroblasts, indicating that it is the subunit of cell-surface fibronectin and identical with the external transformation-sensitive polypeptide of similar molecular weight described by others. Upon coelectrophoresis, the labeled 2.2-10(5) dalton polypeptide migrated slightly behind the subunit of plasma fibronectin (cold-insoluble globulin), indicating that the immunologically cross-reactive forms of fibronectin in human plasma and cultured human fibroblasts differ slightly in molecular weight. The identities of the labeled greater than 1.2-10(6) and 1.3-10(5) dalton polypeptides are not known. The XIIa-reactive glutamine residues of fibroblast cell-surface proteins are potential sites for intermolecular cross-linking (by xi-(gamma-glutamyl)lysyl linkages) to other proteins of connective tissue.     

Sequencing branched peptides with CID/PSD MALDI-TOF in the low-picomole range: application to the structural study of the posttranslational polyglycylation of tubulin

Sequencing conditions for postsource decay and collision-induced dissociation/postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been optimized to elucidate the structure of polyglycylation of tubulin. This posttranslational modification involves the linkage of multiple glycine residues through the gamma-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual alpha- and beta-tubulin polypeptides contain respectively three and four potential glycylation sites. The sample preparation we used was the thin-layer preparation of the target specimen in the presence of alpha-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence DATAEEEGEFEEEGEQ) shows that the peptides fragment regularly to form major fragments of b- and y-type ions with negligible side-chain fragmentation. The rules were applied to the structural elucidation of a Paramecium beta-tubulin hexaglycylated peptide available in the subpicomole range. Polyglycylation was identified on the last four glutamic acid residues.     

Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.     

Development of injectable microcapsules for use in the treatment of narcotic addiction

Injectible microcapsules containing narcotic antagonists have been prepared with dl-poly (lactic acid) as the coating material. The encapsulation technology has developed to the point that high yields of less than 180 mu capsules can be prepared routinely. Such capsules with an initial payload of 50 wt. % naltrexone pamoate provide 60-90% antagonism to the action of morphine 28 days after injection into mice as a peanut oil/aluminum monostearate suspension at a dose level of 40 miligrams naltrexone pamoate/ kg. mouse.