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G Kreil

Publications and source records attributed to G Kreil.

At least 73 records · Page 4Linked to original sources
PubMed1986

Both hydrophobic domains of M13 procoat are required to initiate membrane insertion.

M13 procoat protein has two hydrophobic domains, one in the leader peptide and one which anchors the mature coat protein in the membrane. Disruption of the membrane anchor region by insertion of arginyl residues does not yield periplasmic coat protein. Instead, the rate of membrane assembly is slowed greater than 100-fold (t1/2 less than 5 s for wild-type, t1/2 greater than 10 min for mutant). The hydrophobic region of mature coat protein not only functions as a membrane anchor, but has an important role in the membrane assembly process per se.

Amino Acid Sequence↗
PubMed1986

Isolation of a dipeptidyl aminopeptidase, a putative processing enzyme, from skin secretion of Xenopus laevis.

A dipeptidyl aminopeptidase has been purified to apparent homogeneity from skin secretion of Xenopus laevis. This enzyme is a glycoprotein with a molecular mass of about 98 kDa. It hydrolyzes a variety of dipeptidyl-p-nitroanilides and oligopeptides containing proline, alanine or glycine as the second amino acid and is inhibited by diisopropylfluorophosphate. The pH optimum was found to be around 8, while at pH 6, substrates were cleaved at about one-third of the maximal rate. This dipeptidyl aminopeptidase has the specificity required for the cleavage of amino-terminal extensions preceding the sequence of caerulein and xenopsin in their respective precursors.

Animals↗
PubMed1986

Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis. A small family of precursors containing one, three, or four copies of the final product.

From skin of Xenopus laevis, cDNA libraries were constructed and clones coding for the precursors of caerulein were isolated and sequenced. Using restriction endonuclease digestions, three different types of preprocaerulein cDNAs could be discerned. These were termed types I, III, and IV in accordance with the number of caerulein copies present in the sequence, the type III being the most abundant one. An incomplete copy of a fourth variant, termed type I', was also found. Besides deletions/insertions encompassing one or two caerulein sequences, these types also differ from each other by several point mutations. In the homologous precursor polypeptides deduced from the nucleotide sequence of these cloned cDNAs, the caerulein copies are flanked by complex processing sequences. These are Arg-Arg-Phe-Ala-Asp-Gly or Arg-Arg-Asp-Gly at the amino-terminal side and Gly-Arg-Arg at the carboxyl end. Between caerulein copies, highly homologous segments are present both at the polypeptide and cDNA level. This homology is evident both within a given precursor, where up to three such segments are present, and between the different types of precursors. We conclude that preprocaerulein cDNAs in the skin of X. laevis represent a small family, at least part of which is derived from different genes rather than being formed by alternative splicing of pre-mRNAs.

Amino Acid Sequence↗
PubMed1985

Solid-phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa-(4-24)] from skin secretion of Xenopus laevis.

From the nucleotide sequence of clones isolated from a cDNA library constructed from skin of Xenopus laevis, the existence of PYLa, a peptide comprised of 24 amino acids, was predicted. This peptide was synthesized by solid-phase methods and purified to homogeneity with an overall yield of 61%. The synthetic peptide was used as reference substance to search for its natural counterpart in skin secretion of Xenopus. Two peptides were found which were very similar to PYLa except for the absence of the first three amino acids. These 21-amino-acid peptides, termed PGLa, can be generated from PYLa by cleavage after the single arginine residue present in the latter. The two forms of PGLa differ in their retention time on HPLC but have identical amino acid compositions and terminal sequences. Tryptic hydrolysis of synthetic PYLa after the single arginine yields exclusively PGLa with the shorter retention time on HPLC. The chemical difference between the two forms of PGLa is currently not known. The possible biological role of these newly discovered constituents of frog skin secretion is discussed.

Amino Acids↗
PubMed1985

Biosynthesis of peptides in the skin of Xenopus laevis: isolation of novel peptides predicted from the sequence of cloned cDNAs.

From skin of Xenopus laevis, a few peptides have been isolated which are identical or homologous to gastrointestinal hormones and/or neurotransmitters of mammalian origin. We have studied the biosynthesis of these peptides using recombinant DNA techniques. From cDNA librariers constructed from skin mRNA, clones with inserts coding for the precursors of caerulein, thyrotropin releasing hormone and a new peptide termed PGLa have been isolated and sequenced. In the case of caerulein, a small family of precursors containing one, three or four copies of the end product have been detected. The caerulein sequences are separated by homologous sequences which potentially could give rise to additional constituents of skin secretion. Three such peptides have been detected which are presumably liberated from caerulein precursors by cleavage at single arginine residues.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of cloned cDNAs coding for preprosecapin, a major product of queen-bee venom glands.

Total mRNA from queen-bee venom glands was transcribed into cDNA and cloned into the PstI and ClaI site of pBR322. The nucleotide sequence of two clones (pUM9/24 and pBMC1) with inserts derived from mRNAs for preprosecapin is presented. The two inserts, which are identical in their overlapping regions, have a coding region encompassing 77 triplets. The cloned cDNAs differ in the length of the 3'-untranslated region, which is about twice as long in clone pUM9/24. The postulated amino acid sequence of preprosecapin starts with a signal peptide containing an estimated 32 residues, followed by a pro part which terminates with a single arginine. The end-product secapin is located at the COOH end of the precursor. Activation of prosecapin must, therefore, differ from the processing of promelittin synthesized in the same gland. Cell-free translation of total venom gland mRNA in the presence of radioactive histidine yields only two major products, the smaller of which is presumably preprosecapin. Edman degradation of total translation products yields peaks of radioactive tyrosine and histidine at the 4th and 10th cycles respectively. This confirms the sequence of preprosecapin deduced from the cDNA clones. The content of secapin in queen-bee venom must be higher than it is in worker bee venom.

Amino Acid Sequence↗
PubMed1984

Biosynthesis of thyrotropin releasing hormone in the skin of Xenopus laevis: partial sequence of the precursor deduced from cloned cDNA.

Skin of Xenopus laevis contains relatively large quantities of thyrotropin releasing hormone (TRH). Total mRNA isolated from skin was cloned in the plasmid pUC8. Among 1400 cDNA clones, one was found with an insert of 478 nucleotides coding for the amino-terminal part of prepro-TRH. This clone was detected using a mixture of two synthetic undecanucleotides for colony hybridization. The single open reading frame starts with a methionine residue and a stretch of hydrophobic amino acids, as is typical for signal peptides, and terminates at the poly(C) tail without a stop codon. The deduced polypeptide of 123 amino acids contains three copies of the sequences Lys-Arg-Gln-His-Pro-Gly-Lys Arg-Arg and a fourth incomplete copy at the carboxyl end. Typical pro-hormone processing at this sequence would yield pGlu-His-Pro.NH2,i.e. TRH. It is concluded that the cloned part of the mRNA codes for prepro-TRH and that the TRH precursor from skin of X. laevis is a polyprotein containing at least four copies of the end product in its amino acid sequence.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of cloned cDNA coding for honeybee prepromelittin.

Total mRNA from venom glands of young queen bees was transcribed into cDNA and cloned into the PstI site of plasmid pBR322. The nucleotide sequence of two clones with inserts containing genetic information for prepromelittin is presented. The longer insert encompasses 374 base pairs including 52 nucleotides before the initiation codon and a 3' non-coding region of 112 base pairs. The 70 amino acids of prepromelittin represent the total coding capacity of the mRNA from which this insert is derived. Southern blot analysis with this cloned cDNA has shown that it hybridizes with a single EcoRI fragment of honeybee DNA which contains about 3000 base pairs.

Animals↗
PubMed1983

Biosynthesis of caerulein in the skin of Xenopus laevis: partial sequences of precursors as deduced from cDNA clones.

The decapeptide caerulein represents one of the main constituents of the skin secretion of Xenopus laevis. Total mRNA was isolated from skin, transcribed into cDNA and inserted via GC-tailing into the plasmid pUC8. Among the transformants, 300 clones were selected at random and screened with a cDNA primed with the synthetic deoxynucleotide d(AGTCCATCCA), which is complementary to the mRNA region coding for the fragment Trp-Met-Asp-Phe of cerulein. Of nine strongly hybridizing clones, three were sequenced and these were found to contain inserts with very similar nucleotide sequences. The cloned cDNAs code for parts of two different caerulein precursors. These contain one or two copies of caerulein and five additional amino acids located between pairs of arginine residues. The extra glycine at the carboxy terminus is considered to serve as the signal for amidation, while the tetrapeptide Phe-Ala-Asp-Gly, linked to the amino end of caerulein in these precursors, must be cleaved by an unusual processing mechanism.

Amino Acid Sequence↗
PubMed1983

A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin.

A variety of peptides closely related to mammalian hormones and neurotransmitters are secreted from amphibian skin. Using cDNA clones of mRNA isolated from skin of Xenopus laevis, we have been searching for precursors of some of these constituents. Here we present the sequences of parts of cloned mRNAs which code for precursors of a novel peptide. In the predicted polypeptides, pairs of basic residues flank a sequence of 25 amino acids terminating with glycine, the signal for the formation of a terminal amide. The predicted final product liberated from these precursors would be a peptide comprised of 24 amino acids starting with tyrosine and ending with leucine amide, which has therefore been designated PYLa. This peptide can form an amphipathic helix similar to that found in peptides with cytotoxic, bacteriostatic and/or lytic properties.

Amino Acid Sequence↗
PubMed1983

Studies on the enzymatic degradation of beta-casomorphins.

beta-Casomorphins (beta-CMs), although known to be highly resistant to proteolytic enzymes, are demonstrated to be rapidly degraded in bovine or rat plasma. Degradation of these peptides consisting of the amino acid sequence TYR-PRO-PHE-PRO-GLY-PRO-ILE and C-terminally shortened fragments thereof, may be due to an enzyme identical with or similar to the dipeptidyl-peptidase IV (DP IV) which is known to cleave dipeptide fragments from the N-terminus of peptides after proline residues. This assumption is compatible with the finding that beta-casomorphin (beta-CM) analogues in which the proline residue in position two has been replaced by D-alanine, seem to be completely resistant to enzymatic attack in the plasma.

Amino Acid Sequence↗
PubMed1982

Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide.

It has previously been shown that rat liver microsomes contain a proteolytic enzyme that cleaves honeybee prepromelittin to yield promelittin. This enzyme has now been further purified by centrifugation on a sucrose-deoxycholate gradient and then reconstituted into phospholipid vesicles. Incubation of prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with l-butanol and paper electrophoresis in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. The microsomal enzyme is thus an endoprotease that hydrolyzes prepromelittin exclusively at the pre-pro junction. The precision of this cleavage of an insect preprotein by a rat liver enzyme indicates that we are dealing with the ubiquitous eukaryotic signal peptidase.

Amino Acid Sequence↗
PubMed1981

The sequestration, processing and retention of honey-bee promelittin made in amphibian oocytes.

Messenger RNAs from one kind of secretory cell can be introduced into the cytoplasm of another: the heterologous proteins formed by the recipient cell are usually processed and topologically segregated in the manner characteristic of the donor cell. Xenopus oocytes injected with honey-bee venom gland RNA provide some support for this generalization, but also reveal important exceptions to it. Thus, the frog cell makes a small polypeptide whose partial sequence matches perfectly that of insect promelittin, except that the product formed in oocytes ends at the C terminus with a glycine as opposed to a glutamine amide residue. N-terminal heterogeneity is seen in protoxin made in oocytes and venom gland cells, and species shorter by two residues are seen in both tissues. We suggest that the oocyte contains a dipeptidylpeptidase. Promelittin made in oocytes is barely detectable in the cytosol but is found associated with a vesicle fraction which also contains some newly synthesized endogenous oocyte proteins. The association with vesicles is long-lasting; thus promelittin is retained slightly more efficiently than sequestered oocyte proteins, and an incubation period of about two weeks is required to reduce by half the amount of these endogenous vesicle proteins. Thus neither promelittin nor any products derived from it are secreted rapidly. Gel analysis fails to reveal promelittin in the medium surrounding the oocyte, although traces can be detected by assaying for a characteristic heptapeptide. Such small amounts could result from slow secretion or leakage. Melittin could not be detected by gel analysis or peptide assay. The retention of the honey-bee protein within the frog cell is discussed in terms of the specificity of the processing systems and secretory pathways of venom gland cells and oocytes. We suggest that whilst some export mechanisms function efficiently in a wide variety of cells, others do not, and may even be restricted to specific cell types.

Amino Acid Sequence↗
PubMed1980

Stepwise cleavage of the pro part of promelittin by dipeptidylpeptidase IV. Evidence for a new type of precursor--product conversion.

Melittin, the main constituent of honeybee venom, is derived from promelittin. In the amino acid sequence of the 'pro' region of this precursor, every second residue is either proline or alanine. The possibility has been investigated that activation of promellitin might proceed via sequential liberation of dipeptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2-terminal hexapeptide of melittin. It could first be demonstrated that pig kidney of dipeptidyleptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2-terminal hexapeptide of melittin. It could first be demonstrated that pig kidney of dipeptidylpeptidase IV releases dipeptides from the pro part. An enzyme of this type could then be detected in extracts from venom glands of q bees, which also contain a dipeptidase. After inhibiting the latter enzyme with mersalyl, a stepwise cleavage of dipeptides, starting at the amino end of the pro region could be demonstrated. This hydrolysis did not proceed into the melittin sequence. Furthermore, fragments with an extra residue at the amino end, which therefore had the wrong 'reading frame' for the dipeptidylpeptidase, were not hydrolyzed. With intact promelittin as substrate the rate of hydrolysis was always lower than with the fragments. The results presented in this paper suggest a new type of precursor-product conversion proceeding via stepwise cleavage of dipeptide units. Our experimental evidence also ascribes a biological function to a dipeptidylpeptidase IV, a type of enzyme widely distributed in animals tissues. The evidence, that the observed reaction in vitro reflects the mechanism of promelittin activation in vivo is discussed.

Amino Acid Sequence↗