Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

Degradation of Ovalbumin by L-Ascorbic Acid in the Presence of Cupric Ion

Ovalbumin, L-ascorbic acid and cupric sulfate were allowed to react at pH 3.0, 6.0, 6.8 and 7.5. Non-proteinous nitrogen compounds were formed from ovalbumin coupled with autoxidation of ascorbic acid, and a pronounced increase in their formation was observed in the reactions of neutral pH ranges. Non-proteinous nitrogen compounds contained peptides, free amino acids and ammonia. In the reactions of ovalbumin with triose reductone similar results to those with ascorbic acid were obtained. In the ovalbumin degraded with ascorbic acid at pH 6.8 was found an increase of N-terminal amino acid, which could react with carbonyl compounds resulting in browning.     

Organization of coding and intervening sequences in the chicken ovalbumin split gene

The interruptions in the chicken ovalbumin gene which were reported previously (Breathnach, Mandel and Chambon, 1977) are shown to be due to the presence of intervening sequences which separate the messenger-coding sequences. We present evidence for an additional interruption of the gene, which, together with those reported earlier and by Garapin et al. (1978b), make a total of six intervening sequences. All of these intervening sequences are located in the DNA region that corresponds to the part of the ov mRNA which codes for amino acids. The seven coding fragments of the split ovalbumin gene are arranged in the same order and relative orientation as in the ovalbumin double-stranded cDNA. All the sequences coding for ov mRNA are contained in a chromosomal DNA region of 6000 bp, which is more than 3 times longer than ov mRNA. The general organization of the ovalbumin split gene is discussed.     

Intracellular Distribution of Free Amino Acids between the Vacuolar and Extravacuolar Compartments in Internodal Cells of Chara australis

Distribution of free amino acids between the vacuolar and extra-vacuolar(cytoplasmic) compartments in internodal cells of Chara australiswas studied. Under the control conditions (14-h light : 10-hdark), most (90%) of the cellular free amino acids were foundin the extra-vacuolar compartment. The reverse was true forammonia. The major amino acids were isoasparagine, alanine,glutamic acid, aspartic acid, serine and glycine. The contentsof hydrophobic and basic amino acids were minor and relativelygreater proportions were found in the vacuole except when theircontents were extremely low. When cells were kept for 3 days under continuous light or incontinuous darkness, the total free amino acid content increasedto about 120% (light) and about 150% (dark) that of the control.These increases mainly took place in the vacuole, but the aminoacid species responsible for the increments differed with thelight conditions. In contrast, the cytoplasmic content was relativelyconstant (50–60 mM) even under continuous light or darkness.The results suggest that the vacuole acts in the homeostasisof the cytoplasmic amino acid content. As anion, amino acidsin the cytoplasm compensated for about 10–20% of the reported"anion deficiency" in the cytoplasm. (Received June 7, 1984; Accepted September 11, 1984)     

A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8-11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K(b)-restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODC-LEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K(b) and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides.     

樟芝菌丝体的氨基酸成分分析

测定了樟芝发酵菌丝体中游离氨基酸和结合氨基酸的含量。结果表明,樟芝菌丝体中含有丰富的8种人体必需氨基酸、支链氨基酸和谷氨酸,其中8种必需氨基酸含量是FAO(联合国粮农组织)标准的5.55倍,而芳香族氨基酸的含量却很低。在测定过程中,由于采用了酸水解,菌丝体中的色氨酸被破坏,而其余17种氨基酸均被测出,含量极其丰富。尤其是支链氨基酸的含量远大于α-酪蛋白、卵蛋白和大豆球蛋白。     

Import of frog prepropeptide GLa into microsomes requires ATP but does not involve docking protein or ribosomes.

Frog prepropeptide GLa, a precursor to a secretory protein containing 64 amino acids, was processed and imported by dog pancreas microsomes. These events did not depend on either docking protein or on the presence of ribosomes. A hybrid protein between the first 60 amino acids of prepropeptide GLa and an unrelated peptide of 49 amino acids fused to the carboxy terminus, however, behaved like a typical secretory protein precursor with regard to docking protein dependence. This suggests that independence of the need for docking protein, in the case of prepropeptide GLa, can be attributed to the size of the precursor protein. Processing and import of prepropeptide GLa by microsomes were ATP dependent. Therefore, import of proteins into the endoplasmic reticulum (ER) includes an ATP-requiring step not involving a ribosome/ribosome receptor or signal recognition particle (SRP)/docking protein interaction.     

四个杏鲍菇品种的氨基酸分析与比较

测定了4个杏鲍菇新品种子实体的氨基酸含量,结果表明,4个新品种的子实体中含有丰富的8种人体必需氨基酸、支链氨基酸和带有胍基的精氨酸,而芳香族氨基酸的含量却很低。在测定过程中,由于采用了酸水解,子实体中的色氨酸被破坏,而其余17种氨基酸均被测出,含量极其丰富。尤其是精氨酸、谷氨酸的含量接近于α-酪蛋白、卵蛋白和大豆球蛋白。     

Fine mapping and structural analysis of immunodominant IgE allergenic epitopes in chicken egg ovalbumin

Ovalbumin is a major allergen in hen egg white that causes IgE-mediated food allergic reactions in children. In this study, the immunodominant IgE-binding epitopes of ovalbumin were mapped using arrays of overlapping peptides synthesized on activated cellulose membranes. Pooled human sera from 18 patients with egg allergy were used to probe the membrane. Five distinct regions were found to contain dominant allergic IgE epitopes, these being L38T49, D95A102, E191V200, V243E248 and G251N260. The critical amino acids involved in IgE antibody binding were also determined. These epitopes were composed primarily of hydrophobic amino acids, followed by polar and charged residues and being comprised of beta-sheet and beta-turn structures. One epitope, D95A102, consisted of a single alpha-helix. These results provide useful information on the functional role of amino acid residues to evaluate the structure-function relationships and structural properties of allergic epitopes in ovalbumin. They also provide a strategic approach for engineering ovalbumin to reduce its allergenicity.     

Binding of truncated peptides to the MHC molecule IA (d).

A peptide comprising amino acids 323-339 of chicken ovalbumin is known to bind to two heterodimeric conformations of the MHC molecule IA(d), and to each of its separate alpha- and beta-chains. We report that minor C- and N-terminal truncations of the parent peptide do not alter the binding pattern. A decrease in binding activity was observed upon deletion of the histidine residues of the already truncated peptides. Peptides as short as 4 amino acids associate weakly with all four proteins.     

Sugar, Amino Acid and Inorganic Contents in Rice Phloem Sap

Rice phloem sap was obtained through severed stylets of brownplanthoppers and its chemical composition was determined. Sucrose,the only sugar detected, was present at 17–25% (w/v) inthe sap. Amino acids were present at a total of 3–8% (w/v)and were found to be mostly in a free form. The amount of boundamino acids was estimated to be small, if any were present.Among the free amino acids, asparagine (17–33% on a molarbasis), glutamate (6–14%), serine (10–13%), glutamine(7–15%), threonine (5–6%) and valine (6–7%)were dominant, while cystine and methionine (0–0.2%) werepresent in minor amounts and -aminobutyric acid was not detected.The sap had a slightly alkaline pH (ca. 8.0). The inorganicconstituents detected by electron probe x-ray microanalyzerwere Na, S, P and K, with the K content being the highest. Theosmotic pressure was estimated to be 13–15 atm. The aminoacid composition of the plant parts was determined and the differencesin the ease of phloem loading among amino acids were compared. (Received October 5, 1981; Accepted December 17, 1981)     

Nucleotide sequence at the 5'' end of ovalbumin messenger RNA from chicken.

DNA-sequence analysis of 300 nucleotides from the region of cloned, double-stranded ovalbumin cDNA corresponding to the 5' end of ovalbumin messenger RNA was accomplished using the technique of Maxam and Gilbert (Proc. Nat. Acad. Sci. USA (1977) 74,560-564). The AUG initiation codon was located 52 nucleotides from the AT linkers used in cloning and immediately adjacent to the amino terminal peptide of ovalbumin, indicating the absence of a "signal peptide" in this protein. The nucleotide sequence coding for a phosphorylated peptide from ovalbumin was also found. These results demonstrate that the coding portion of mRNAov begins near the 5' end of the molecule leaving some 600 nucleotides of noncoding information at the 3' end.     

Import of honeybee prepromelittin into the endoplasmic reticulum: energy requirements for membrane insertion.

The import of small precursor proteins, derived from the honeybee secretory protein prepromelittin, into dog pancreas microsomes is independent of signal recognition particle (SRP) and docking protein, but requires that charged amino acids at the amino terminus of the mature part are counterbalanced by amino acids with the opposite charge at the carboxy terminus. The import pathway of such precursor proteins was resolved into two sequential steps: (i) binding of precursors to microsomes, and (ii) insertion of precursors into the membrane. Formation of an intramolecular disulfide bridge within the mature part of these precursor proteins allowed association of the oxidized precursors with the microsomal membrane but reversibly inhibited their membrane insertion. Furthermore, membrane insertion was inhibited by ATP depletion. Different prepromelittin derivatives were found to depend on ATP to varying degrees. We conclude that insertion of prepromelittin-derived precursor proteins into microsomal membranes involves a competent conformation of the precursor proteins and that, in general, this is accomplished with the help of both a cytoplasmic component and ATP.     

Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein.

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles.     

Characterization of beta 1----4 galactosyltransferase purified from rat liver microsomes.

beta 1----4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound beta 1----4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53,000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse beta 1----4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the NH2-terminal 12 amino acids from a precursor polypeptide.     

Flower-promoting effect of some amino acids and amides in Lemna paucicostata 6746

Cultures of Lemna paucicostata 6746 were exposed to a single96-hr dark period followed by continuous illumination at 24?1?C.Flowering percentage increased to a maximum 3 days after theend of the dark period and then fell off to 0% on the 5th day.Among 20 amino acids and 2 amides tested, addition of asparagine,aspartate, glutamate, -alanine, glycine and serine clearly increasedthe flowering percentages and retarded the regression of floralbuds by 2–3 days. These substances given after the endof the long dark period were more effective than those givenduring the dark period, suggesting that they favored the flower-producingprocess following the inductive dark process. On the other hand,if the above amino acids or amide were applied under repeatedlight-dark cycles, they shortened the critical dark period by1–2 hr and almost completely nullified the light-breakeffect. They seem to promote the flower-inductive dark process,too. Glutamate, for instance, was effective even at 5 µM, whilethis amino acid is found in the plant body in large quantities.The mechanism of flower promotion by these amino acids and amideremains unknown. (Received June 3, 1976; )     

AUTORADIOGRAPHIC ANALYSIS ON AGAR PLATES OF ANTIGENS FROM SUB CELLULAR FRACTIONS OF RAT LIVER SLICES

Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of ¹⁴C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens.     

The ovalbumin gene family: complete sequence and structure of the Y gene.

The "ovalbumin Y" gene, one of three which constitute the ovalbumin gene family in chicken has been completely sequenced. The exact location of exons can be derived from the comparison with the ovalbumin gene sequence and from the map previously established by electron microscopy analysis. During evolution of the Y gene, selective pressure has operated to retain a sequence coding for an ovalbumin-like protein. The location of splice junctions, the length of protein coding exons and the reading phase are as in the ovalbumin gene. The overall homology between the Y and ovalbumin protein coding sequences is 72.6% (resulting in a 58% homology for the amino acid sequences). A significantly high number of base changes within coding sequences are present in clusters, which appear in several cases to be correlated with the occurrence of direct repeats. The 3' untranslated sequences of the Y and ovalbumin mRNAs have diverged much more, and the Y sequence contains a peculiar U(T) rich region. Corresponding introns of the ovalbumin and Y genes differ extensively both in sequence and in length. They share however characteristic biases in their base distribution.