Left-handed polyproline II helices (PPII) are contiguous elements of protein secondary structure in which the phi and psi angles of constituent residues are restricted to around -75 degrees and 145 degrees, respectively. They are important in structural proteins, in unfolded states and as ligands for signaling proteins. Here, we present a survey of 274 nonhomologous polypeptide chains from proteins of known structure for regions that form these structures. Such regions are rare, but the majority of proteins contain at least one PPII helix. Most PPII helices are shorter than five residues, although the longest found contained 12 amino acids. Proline predominates in PPII, but Gln and positively charged residues are also favored. The basis of Gln's prevalence is its ability to form an i, i + 1 side-chain to main-chain hydrogen bond with the backbone carbonyl oxygen of the proceeding residue; this helps to fix the psi angle of the Gln and the phi and psi of the proceeding residue in PPII conformations and explains why Gln is favored at the first position in a PPII helix. PPII helices are highly solvent exposed, which explains why apolar amino acids are disfavored despite preferring this region of phi/psi space when in isolation. PPII helices have perfect threefold rotational symmetry and within these structures we find significant correlation between the hydrophobicity of residues at i and i + 3; thus, PPII helices in globular proteins can be considered to be amphipathic.

Actin is the principal constituent of the thin filaments of muscle, and in order to provide information basic to understanding the molecular basis of actin function we have studied its amino acid sequence. The isolation, compositions, and sequences of cyanogen bromide peptides, ranging in size from 3 to 44 residues, have previously been reported (ELZINGA, M. (1971) Biochemistry 10, 224-229, and other papers in the present series). The peptides have been aligned by isolation and characterization of tryptic peptides that contain methionine. The isolation of one of the CNBr peptides (CB-14) was complicated by the presence of a Met-Thr bond that was only partially split under standard conditions for cyanogen bromide cleavage in formic acid. In this paper conditions are described for increasing the cleavage at this bond. CB-14 is a tetrapeptide, Thr-Gln-Ile-Hse, and this sequence completes the characterization of the actin cyanogen bromide peptides. Finally, the position of CB-14 in the actin sequence as residues 120 to 123 was established by isolation of a chymotryptic overlap peptide. The complete sequence of the 374 residues of actin is presented.

A specific antiviral cytotoxic immune response in vivo could be induced by the subcutaneous injection of the T-cell epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein as an unmodified free synthetic peptide (Arg-Pro-Gln-Ala-Ser-Gly-Val-Tyr-Met-Gly-Asn-Leu-Thr-Ala-Gln) emulsified in incomplete Freund's adjuvant. This immunization rendered mice into a LCMV-specific protective state as shown by the inhibition of LCMV replication in spleens of such mice. The protection level of these mice correlated with the ability to respond to the peptide challenge by CD8+ virus-specific cytotoxic T cells. This is a direct demonstration that peptide vaccines can be antivirally protective in vivo, thus encouraging further search for appropriate mixtures of stable peptides that may be used as T-cell vaccines.

Putative high-affinity nitrate (NO3-) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by RT-PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30-73% identities with other eukaryotic high-affinity NO3- transporters. Both genes were induced by NO3-, but Nrt2;1At gene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter, Nrt2;1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression levels were strongly correlated with inducible high-affinity 13NO3- influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation of Nrt2;1At expression was mediated by NH4+, gln and other amino acids.

Obesity is associated with multiple adverse health effects and a high risk of developing metabolic and cardiovascular diseases. Therefore, there is a great need to identify circulating parameters that link changes in body fat mass with obesity. This study combines proteomic and metabolomic approaches to identify circulating molecules that discriminate healthy lean from healthy obese individuals in an exploratory study design. To correct for variations in physical activity, study participants performed a one hour exercise bout to exhaustion. Subsequently, circulating factors differing between lean and obese individuals, independent of physical activity, were identified. The DIGE approach yielded 126 differentially abundant spots representing 39 unique proteins. Differential abundance of proteins was confirmed by ELISA for antithrombin-III, clusterin, complement C3 and complement C3b, pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP4), serum amyloid P (SAP), and vitamin-D binding protein (VDBP). Targeted serum metabolomics of 163 metabolites identified 12 metabolites significantly related to obesity. Among those, glycine (GLY), glutamine (GLN), and glycero-phosphatidylcholine 42:0 (PCaa 42:0) serum concentrations were higher, whereas PCaa 32:0, PCaa 32:1, and PCaa 40:5 were decreased in obese compared to lean individuals. The integrated bioinformatic evaluation of proteome and metabolome data yielded an improved group separation score of 2.65 in contrast to 2.02 and 2.16 for the single-type use of proteomic or metabolomics data, respectively. The identified circulating parameters were further investigated in an extended set of 30 volunteers and in the context of two intervention studies. Those included 14 obese patients who had undergone sleeve gastrectomy and 12 patients on a hypocaloric diet. For determining the long-term adaptation process the samples were taken six months after the treatment. In multivariate regression analyses, SAP, CLU, RBP4, PEDF, GLN, and C18:2 showed the strongest correlation to changes in body fat mass. The combined serum proteomic and metabolomic profiling reveals a link between the complement system and obesity and identifies both novel (C3b, CLU, VDBP, and all metabolites) and confirms previously discovered markers (PEDF, RBP4, C3, ATIII, and SAP) of body fat mass changes.

The mechanisms involved in regulating high-affinity ammonium (NH4+) uptake and the expression of the AtAMT1 gene encoding a putative high-affinity NH4+ transporter were investigated in the roots of Arabidopsis thaliana. Under conditions of steady-state nitrogen (N) supply, transcript levels of the AtAMT1 gene and Vmax values for high-affinity 13NH4+ influx were inversely correlated with levels of N provision. Following re-supply of NH4NO3 to N-starved plants, AtAMT1 mRNA levels and 13NH4+ influx declined rapidly but remained high when the conversion of NH4+ to glutamine (Gln) was blocked with methionine sulfoximine (MSX). This result demonstrates that end products of NH4+ assimilation, rather than NH4+ itself, are responsible for regulating AtAMT1 gene expression. Consistent with this hypothesis, AtAMT1 gene expression and NH4+ influx were suppressed by provision of Gln alone, or together with NH4NO3 plus MSX. Furthermore, AtAMT1 transcript levels and 13NH4+ influx were negatively correlated with root Gln concentrations, following re-supply of N to N-starved plants. In addition to this level of control, the data suggest that high cytoplasmic [NH4+] may inhibit NH4+ influx.

Mammalian nutrient sensors are novel targets for therapeutic intervention in disease states such as insulin resistance and muscle wasting; however, the proteins responsible for this important task are largely uncharacterized. To address this issue we have dissected an amino acid (AA) sensor/effector regulon that controls the expression of the System A amino acid transporter SNAT2 in mammalian cells, a paradigm nutrient-responsive process, and found evidence for the convergence of at least two sensor/effector pathways. During AA withdrawal, JNK is activated and induces the expression of SNAT2 in L6 myotubes by stimulating an intronic nutrient-sensitive domain. A sensor for large neutral AA (e.g. Tyr, Gln) inhibits JNK activation and SNAT2 up-regulation. Additionally, shRNA and transporter chimeras demonstrate that SNAT2 provides a repressive signal for gene transcription during AA sufficiency, thus echoing AA sensing by transceptor (transporter-receptor) orthologues in yeast (Gap1/Ssy1) and Drosophila (PATH). Furthermore, the SNAT2 protein is stabilized during AA withdrawal.

The X-ray structure of a canonical GCN5-related N-acetyltransferase (GNAT), Serratia marcescens aminoglycoside 3-N-acetyltransferase, bound to coenzyme A (CoA) has been determined at 2.3 A resolution. The single domain alpha/beta protein resembles a cupped right hand wrapped around a cylinder and consists of a highly curved, six-stranded beta sheet of mixed polarity that is sandwiched between four alpha helices. The structure includes all four conserved GNAT motifs (C, D, A, and B) and represents the catalytic core of this large enzyme superfamily. Acetyl CoA recognition is mediated by a betaalpha structure derived from GNAT motif A, which presents an invariant Arg/Gln-X-X-Gly-X-Gly/Ala segment for hydrogen bonding with the cofactor. Motif B contributes acidic residues to the binding site for the positively charged antibiotic substrate.

BACKGROUND

Polymorphisms in DNA repair genes have been associated to repair DNA lesions, and might contribute to the individual susceptibility to develop different types of cancer. Nucleotide excision repair (NER), base excision repair (BER), and double-strand break repair (DSBR) are the main DNA repair pathways. We investigated the relationship between polymorphisms in two NER genes, XPC (poly (AT) insertion/deletion: PAT-/+) and XPD (Asp312Asn and Lys751Gln), the BER gene XRCC1 (Arg399Gln), and the DSBR gene XRCC3 (Thr241Met) and the risk of developing lung cancer.

METHODS

A hospital-based case-control study was designed with 516 lung cancer patients and 533 control subjects, matched on ethnicity, age, and gender. Genotypes were determined by PCR-RFLP and the results were analysed using multivariate unconditional logistic regression, adjusting for age, gender and pack-years.

RESULTS

Borderline association was found for XPC and XPD NER genes polymorphisms, while no association was observed for polymorphisms in BER and DSBR genes. XPC PAT+/+ genotype was associated with no statistically significant increased risk among ever smokers (OR = 1.40; 95%CI = 0.94-2.08), squamous cell carcinoma (OR = 1.44; 95%CI = 0.85-2.44), and adenocarcinoma (OR = 1.72; 95%CI = 0.97-3.04). XPD variant genotypes (312Asn/Asn and 751Gln/Gln) presented a not statistically significant risk of developing lung cancer (OR = 1.52; 95%CI = 0.91-2.51; OR = 1.38; 95%CI = 0.85-2.25, respectively), especially among ever smokers (OR = 1.58; 95%CI = 0.96-2.60), heavy smokers (OR = 2.07; 95%CI = 0.74-5.75), and adenocarcinoma (OR = 1.88; 95%CI = 0.97-3.63). On the other hand, individuals homozygous for the XRCC1 399Gln allele presented no risk of developing lung cancer (OR = 0.87; 95%CI = 0.57-1.31) except for individuals carriers of 399Gln/Gln genotype and without family history of cancer (OR = 0.57; 95%CI = 0.33-0.98) and no association was found between XRCC3 Thr241Met polymorphism and lung cancer risk (OR = 0.92; 95%CI = 0.56-1.50), except for the 241Met/Met genotype and squamous cell carcinoma risk (OR = 0.47; 95%CI = 0.23-1.00).

CONCLUSIONS

In conclusion, we analysed the association between XPC, XPD, XRCC1, and XRCC3 polymorphisms and the individual susceptibility to develop lung cancer in the Spanish population, specifically with a highly tobacco exposed population. We attempt to contribute to the discovery of which biomarkers of DNA repair capacity are useful for screening this high-risk population for primary preventing and early detection of lung cancer.

Rhodopsin, the dim light photoreceptor of the rod cell, is an integral membrane protein that is glycosylated at Asn-2 and Asn-15. Here we report experiments on the role of the glycosylation in rhodopsin folding and function. Nonglycosylated opsin was prepared by expression of a wild-type bovine opsin gene in COS-1 cells in the presence of tunicamycin, an inhibitor of asparagine-linked glycosylation. The non-glycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (lambda max, 500 nm) with 11-cis-retinal. However, the nonglycosylated rhodopsin showed strikingly low light-dependent activation of GT at concentration levels comparable with those of glycosylated rhodopsin. Amino acid replacements at positions 2 and 15 and the cognate tripeptide consensus sequence [Asn-2->>Gln, Gly-3->>Cys(Pro), Thr-4->>Lys, Asn-15->>Ala(Cys, Glu, Lys, Gln, Arg), Lys-16->>Cys(Arg), Thr-17->>Met(Val)] showed that the substitutions at Asn-2, Gly-3, and Thr-4 had no significant effect on the folding, cellular transport, and/or function of rhodopsin, whereas those at Asn-15 and Lys-16 caused poor folding and were defective in transport to the cell surface. Further, mutant pigments with amino acid replacements at Asn-15 and Thr-17 activated GT very poorly. We conclude that Asn-15 glycosylation is important in signal transduction.

A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Expansion of the long (CAG; glutamine)n repeat in the first exon of the X-linked human androgen receptor gene (hAR) causes spinal and bulbar muscular atrophy, frequently in association with mild androgen insensitivity. The relevant normal motor neurons are preferentially stimulated by androgen, however no motor neuron disorder occurs with any other known AR mutation, including those that cause complete androgen insensitivity. We have found that a polyglutamine (Gln) expanded AR transactivates an androgen-responsive reporter gene subnormally. Other groups have reported that a poly Gln-deleted AR transactivates normally. A parsimonious interpretation of all these facts is that poly Gln expansion causes the AR to lose a function that is necessary for full androgen sensitivity and to gain a function that is selectively motor neuronotoxic.

To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.

Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor. The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein. All known or predicted cleavage sites in the TEV polyprotein are flanked by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Gly dipeptide. By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site. The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase. Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis. Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.

Proinsulin in nearly homogeneous form has been isolated from a preparation of porcine insulin. A molecular weight close to 9100 was calculated from the amino acid composition and from sedimentation-equilibrium studies. Through the action of trypsin this single-chain protein is transformed to desalanine insulin by cleavage of a polypeptide chain connecting the carboxy-terminus of the B chain to the amino-terminus of the A chain of insulin. The amino acid sequence of this connecting peptide was found to be Arg-Arg-Glu-Ala-Gln-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-Arg.

Cysteine proteases related to mammalian interleukin-1 beta-converting enzyme (ICE) and the nematode cell death abnormal ced-3 gene product have been implicated in the effector mechanism of apoptotic cell death. Two novel members of this new family of ICE/CED-3-related proteases, designated ICErel-II and ICErel-III, were cloned from human monocytic cells. Both were highly homologous to human ICE (52% identical) and CED-3 (25% identical) and both contained the absolutely conserved pentapeptide sequence Gln-Ala-Cys-Arg-Asp containing the catalytic cysteine residue. Other structural motifs that were comparable with ICE suggest that ICErel-II and ICErel-III are also synthesized as larger proenzymes which are proteolytically processed to form heterodimeric active enzymes. Pro-interleukin-1 beta processing activity could not be detected in cells transfected with ICErel-II or ICErel-III, but pro-domain-less truncated forms of ICErel-II and ICErel-III were capable of effectively inducing fibroblast apoptosis. ICErel-II and ICErel-III may, therefore, participate in proteolytic events culminating in the apoptotic death of human cells.

Matrix metalloproteinase 7 (MMP-7) has been purified as an inactive zymogen of M(r) 28,000 (proMMP-7) from the culture medium of CaR-1 human rectal carcinoma cells. The NH2-terminal sequence of proMMP-7 is Lys-Pro-Lys-Pro-<em>Gln</em>-Glu, which is identical to that of matrilysin. The zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding an intermediate form of M(r) 21,000 and an active species of M(r) 19,000 which shows the new NH2-terminal sequence of Tyr78-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully activates the zymogen, the activation rate by plasmin or leukocyte elastase is confined to approximately 50%. ProMMP-7 can be activated by MMP-3 (stromelysin 1) to its full activity in a single-step mechanism and generates the same NH2 terminus obtained by APMA activation, whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9 (gelatinase B) do not have such an effect. On the other hand, proMMP-1 is activated by MMP-7 to an activity similar to that obtained by APMA and the activation by MMP-7 is enhanced up to approximately 6.5 fold in the presence of APMA. This enhanced activity is donated by specific cleavage at the <em>Gln</em>80-Phe81 bond of proMMP-1. MMP-7 can also activate proMMP-9 up to approximately 50% of the full activity with a new NH2 terminus of Leu16-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3 with MMP-7 results in no activation of these proMMPs. MMP-7 degrades type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin, and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix macromolecules in concert with MMP-1, -3, and -9 under pathological conditions.

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.

Two peptides that crossreact with an antiserum raised against Phe-Met-Arg-Phe-NH2 were purified from bovine brain extract. Their structures were determined to be Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 and Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2. The sequences were determined by gas-phase sequencing, except for the COOH-terminal phenylalaninamides. These were assigned on the basis of the reactivity of the peptides with the anti-Phe-Met-Arg-Phe-NH2 antiserum, which appears to recognize the determinant -Arg-Phe-NH2. Both peptides were synthesized, and the synthetic peptides were found to have the same HPLC retention times as the endogenous Phe-Met-Arg-Phe-NH2-immunoreactive peptides, thus confirming the assignment of phenylalaninamide to the COOH-terminal positions. Both of the synthetic peptides were found to decrease tail-flick latency in rats, and the octapeptide was more active than the octadecapeptide. The octapeptide was found also to attenuate the prolongation of the tail-flick latency induced by morphine.

This article describes the fundamental cleavage reactions of (M-H)(-) anions of underivatized peptides that contain up to 25 amino acid residues. The experimental observations of these cleavages have been backed up by molecular modeling, generally at the AM1 level of theory. The basic cleavages are the ubiquitous alpha- and beta-backbone cleavage reactions, which provide information similar to that of the B and Y + 2 cleavages of MH(+) ions of peptides. The residues Asp and Asn also effect cleavages of the backbone (called delta- and gamma-cleavages), by reactions initiated from side chain enolate anions, causing elimination reactions that cleave the backbone between the Asp (Asn) N bond;C backbone bond. Glu and Gln also direct analogous delta- and gamma-cleavages of the backbone, but in this case the processes are initiated by attack of the side chain CO(2) (-) (CONH(-)) to form a lactone (lactam). Ser and Thr residues undergo characteristic fragmentations of the side chain. These processes, losses of CH(2)O (Ser) and MeCHO (Thr), convert these residues into Gly. In larger peptides, Ser and Thr can effect two backbone cleavage reactions, called gamma- and epsilon -processes. The C-terminal CO(2) (-) (or CONH(-)) forms a hydrogen bond with the side chain OH (of Ser or Thr), placing the C-terminal residue in a position where it may affect S(N) (2) attack at the electrophilic backbone CH of Ser, with concomitant cleavage of the backbone. All of the above negative ion cleavages require the peptide backbone to be conformationally flexible. However, there is a backbone cleavage that requires the peptide to have an alpha-helical conformation in order for the two reacting centers to approach. This cleavage is illustrated for the Glu 23-initiated backbone cleavage at Ile 21 for the (M-H)(-) anion of the antimicrobial peptide caerin 1.1.

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a beta-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.

Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.