Comparison of the antigenic peptides between human prostatic and lysosomal acid phosphatases

The relative antigenicity (capacity to bind antibodies raised against the intact prostatic acid phosphatase) of the selected peptides from human prostatic and lysosomal acid phosphatases was evaluated in a competitive assay. Both prostatic and lysosomal acid phosphatases were shown to possess similar antigenic determinants on both terminal regions, along with more similarity on NH₂-terminal peptide than COOH-terminal site. At least one additional antigenic site is present at the internal region of prostatic acid phosphatase, since the mixture of both amino- and carboxyl-terminal peptides exhibited only 70% inhibition.     

The paramyxovirus fusion protein forms an extremely stable core trimer: structural parallels to influenza virus haemagglutinin and HIV-1 gp41.

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit F2 and a membrane anchored subunit F1. A highly stable structure has been identified comprised of peptides derived from the simian virus 5 (SV5) F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the SV5 F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm > 90 degrees C), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 M(r)) that is resistant to denaturation by 2% SDS at 40 degrees C. The authors suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that is either fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptide N-1 inhibits cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41 and influenza virus haemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.     

NUTRITIONAL STUDIES OF TWO RED ALGAE. II. KINETICS OF AMMONIUM AND NITRATE UPTAKE1, 2

Similar NH₄₊ and NO₃^?.uptake kinetic patterns were observed in Neoagardhiella baileyi (Harvey ex Kiitzing) Wyinne & Taylor and Gracilaria foliifera (Forssk?l) Borgesen. NO₃^? was taken up in a rate-sturating fashion described by the Michaelis-Menten equation. NH₄₊ uptake was multicomponent: a saturable component was accompanied by a diffusive or a high K component showing no evidence of saturation (at ≤50 μM [NH₄₊]). Nitrogen starved plantsi(C/N atom ratios > ca. 10) showed higher transient rates of NH₄₊ uptake at a given concentration than plants not N-Iimited. Only plants with high N content exhibited diel changes inNH₄₊ uptake rates, and showed transient rates of NH₄₊ accumulation which did not greatly exceed the capacity to incorporate N in steady-state growth. NH₄₊ was preferred over NO_3?even in plants preconditioned on NO_3?as the sole N. source, NO_3? uptake was suppressed at 5μM [NH₄₊], but simultaneous uptake occurred at unsurpressed rates at lower concentrations. Potential for N accumulation was greater via NH₄₊uptake than via NO_3?uptake. Changing capacity for NH₄₊ uptake with N content appears to be a mechanism whereby excessive accumulation of N was avoided by N-.satiated plants but a large accumulation was possible for N-depleted plants.     

The phytochromes: A biochemical mechanism of signaling in sight?

The biochemical mechanism by which the phytochrome family of plant sensory photoreceptors transmit perceived informational light signals downstream to transduction pathway components is undetermined. The recent sequencing of the entire genome of the cyanobacterium Synechocystis, however, has revealed a protein that has an NH₂-terminal domain with striking sequence similarity to the photosensory NH₂-terminal domain of the phytochromes, and a COOH-terminal domain strongly related to the transmitter histidine kinase module of bacterial two-component sensors. The Synechocystis protein is capable of autocatalytic chromophore ligation and exhibits photoreversible light-absorption changes analogous to the phytochromes, indicating its capacity to function as an informational photoreceptor. Together with earlier observations that the COOH-terminal domains of the plant phytochromes also have sequence similarity to the histidine kinases, these data suggest that the cyanobacteria utilize photoregulated histidine kinases as a sensory system and that the plant phytochromes may be evolutionary descendants of these photoreceptors.     

A simulation model for the effect of predation on bacteria in continuous culture

A simulation model was developed for the carbon (C), nitrogen (N), and phosphorus (P) content of bacteria and their medium in a chemostat. Cell components distinguished included the structural component, synthetic machinery, building blocks and intermediates, C reserves, ammonium (NH₄), orthophosphate (PO₄), and polyphosphate. Growth, incorporation of substrates, and production of waste products were related to physiological status, as indicated by the amounts of various cell components. The model was fitted to data from chemostats on the chemical composition of bacteria growing in C-, N-, and P-limiting media and was used to explore the consequences of predation on bacterial populations. In C-limiting media predation (without the return of nutrients to the medium by the predator) increased NH₄ uptake in spite of a decrease in bacterial biomass. In N-limiting media predation decreased both biomass and the rate of N uptake. These results were accounted for by the effect of growth rate on bacterial N demand. In C-limiting media the return of NH₄ and PO₄ by the predator did not change the effect of predation on bacteria. But in N-limiting media the return of nutrients decreased the effect of predation on biomass, and stimulated respiration and NH₄ uptake by the bacteria. The effect of growth rate on the chemical composition of bacteria was proposed as a possible explanation of the stimulatory effect of predators on bacteria.     

Orientation of the cleavage map of the 200-kilodalton polypeptide encoded by the bottom-component RNA of cowpea mosaic virus

The genomic organization of the bottom-component RNA of cowpea mosaic virus was studied. In vivo, this RNA encodes at least eight different polypeptides of 170, 110, 87, 84, 60, 58, 32, and 4 kilodaltons (K), the last polypeptide representing the genome-bound protein VPg. In rabbit reticulocyte lysates, bottom-component RNA is translated into a 200K polypeptide which is then processed to give the 32 and 170K polypeptides also found in vivo. By pulse-labeling the 200K primary translation product, we now show that the 32 and 170K polypeptides are derived from the NH₂-terminal and COOH-terminal parts of this polypeptide, respectively. Comparison of the proteolytic peptide patterns of 170K polypeptides synthesized in vitro and pulse-labeled at either the NH₂-terminal or the COOH-terminal end with the patterns of the 170 and 110K polypeptides found in vivo demonstrates that the order within the 200K primary translation product of cowpea mosaic virus bottom-component RNA is as follows: NH₂-32K polypeptide-58K polypeptide-VPg-24K polypeptide-87K polypeptide-COOH.     

An emerging molecular map of the phytochromes

Molecular mapping studies and sequence comparisons are providing provocative new insights into regions of the phytochrome polypeptide important to the functional activities of the photoreceptor. The NH₂-terminal structural domain contains the determinants for photoperception, and for the differences in photosensory specificity and photolability between phyA and phyB. However, a contiguous COOH-terminal domain is also required for the transfer of perceived informational signals downstream to transduction pathway components and for PfrA-specific degradation to proceed. The COOH-terminal domains of phyA and phyB are functionally interchangeable in these processes and a core sequence at the proximal end of this domain contains determinants necessary for signal transfer from both phyA and phyB, suggesting a common biochemical mechanism of signal transfer for the two photoreceptors. Striking sequence similarity between the NH₂-terminal domain of a Synechocystis protein, ORF SLR0473, and the phytochromes indicates that the cyanobacteria contain phytochrome-related photoreceptors. The COOH-terminal domains of ORF SLR0473 and the phytochromes are also related to one another and both show sequence similarities to the sensor histidine kinases. These data raise the possibility that the cyanobacteria have a functional photoregulated histidine kinase signalling system and that the plant phytochromes represent remnants of that system.     

Studies on the structure of rabbit muscle aldolase: Amino acid sequence of cysteine-containing peptides

Five cysteine-containing peptides have been isolated in nearly stoichemometric yields from the tryptic digests of the NH₂^? and COOH-terminal BrCN peptides of rabhit muscle aldolase and their sequence determined. Peptides NS1, NS2, and NS3, from the NH₂-terminal part of the enzyme have the following sequences: NS1, Val-Asp-Pro-Cys-Ile-Gly-Gly-Val-Ile-Leu-Phe-His-Glu-Thr-Leu-Tyr-Gln-Lys; NS2, Cys-Val-Leu-Lys; NS3, Cys-Ala-Glu-Tyr-Lys. The two peptides isolated from the COOH-terminal region are: CS1, Ala-Leu-Ala-Asn-Ser-Leu-Ala-Cys-Gln-Gly-Lys and CS2, Cys-Pro-Leu-Leu-Trp-Pro-Lys-Ala-Leu-Thr-Phe-Ser-Tyr-Gly-Arg. The Lys-Ala bond in peptide CS2 was found to be resistant to tryptic hydrolysis. The results provide the basis for assigning the positions of cysteine residues in the polypeptide chain. Cys-72 in peptide NS1 and Cys-336 in peptide CS1 are the residues that form a disulfide bridge when the enzyme is inactivated by oxidation with an o-phenanthroline-Cu²⁺ complex; Cys-287 in peptide CS2 in one of the two exposed residues, while Cys-134 and Cys-149 in peptides NS2 and NS3, respectively, are buried in the native enzyme. All of eight cysteine-containing peptides of rabbit muscle aldolase have now been sequenced, and structural homology of the α and β subunits extended to these regions.     

Covalent binding of the natural antimicrobial peptide indolicidin to DNA abasic sites

Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH₂ groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides.     

Guinea pig acylphosphatase: The amino acid sequence

We determined the primary structure of guinea pig skeletal muscle acylphosphatase, using the high degree of homology with several vertebrate acylphosphatases to obtain correct alignment of the complete series of tryptic peptides. Their sequences were obtained mainly by Edman degradation; FAB mass spectrometry was used to identify the acyl group blocking the NH₂-terminal residue and to elucidate the structure of the NH₂-terminal tryptic peptide. The comparison among acylphosphatase sequences from skeletal muscle of several vertebrate species is presented and discussed.     

L-Arginine identified as an endogenous activator for soluble guanylate cyclase from neuroblastoma cells

Guanylate cyclase in neuroblastoma N1E 115 cells was readily solubilized upon homogenization of the cells with hypotonic buffer. When the supernatant was passed through cation exchangers such as a Chelex 100 Na+ column, the guanylate cyclase activity in the effluent fraction decreased to 4-6% of the original supernatant. The addition of the acid extract of neuroblastoma cells or rat tissues to the effluent restored guanylate cyclase activity, indicating that the supernatant of neuroblastoma cells contained an acid-soluble endogenous activator for guanylate cyclase which was adsorbed on cation exchangers. The activator was purified from rat brain and identified as L-arginine by 13C- and 1H-NMR spectroscopy and paper partition chromatography. L-Arginine, at a concentration of 1-2 x 10(-5) M, stimulated guanylate cyclase activity in the effluent fraction 15-25-fold, whereas D-arginine and other basic L-amino acids were ineffective. Peptides that contained L-arginine at the NH2- or COOH-terminal also resulted in an activation of guanylate cyclase to the extent similar to that of L-arginine, while peptides that contained L-arginine inside the peptide chain failed to stimulate the activity. The activation of L-arginine seemed to operate by a mechanism similar to that induced by nitroso compounds.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.     

Cross-Linking of Fibronectin to C-Terminal Fragments of the Fibrinogen α-Chain by Factor XIIIa

Fibronectin binds specifically to fibrin and is covalently cross-linked to the fibrin α chain by activated factor XIII (XIIIa). This reaction is important for wound healing. Here we investigate XIIIa-catalyzed cross-linking of fibronectin and some of its fragments to a recombinant fragment representing the COOH-terminal 30kDa of the fibrin α chain (αC30K:His 368–Val 610). Only fibronectin and those fragments containing an intact NH₂-terminus were able to form cross-linked complexes. As many as 10 of the 17 lysines in αC30K can serve as amine donors in this reaction. Analysis of the rate of XIIIa-catalyzed cross-linking of fibronectin NH₂-terminal peptides and fragments with αC30K revealed that the presence of the first type I “finger” module accelerates the cross-linking reaction; addition of fingers 2–5 had no further effect.     

Study of the primary structures of the peptide core of bovine estrus cervical mucin. Possible existence of small similar subunits

Two populations of tryptic peptides were isolated from bovine estrus cervical mucin (BCM). One contained all the carbohydrate, and was rich in threonine and serine. These glycopeptides had, like the whole mucin, alanine as their NH2-terminal residues. Their COOH-terminal residues were arginine. The second population of peptides was rich in carboxylic amino acids, contained two cysteinyl residues, and had, like the whole mucin, leucine as COOH-terminal residues. Their NH2-terminal residues were aspartic acid. The sum of the residues of one glycopeptide plus one cysteinyl-containing peptide corresponded to the number of residues constituting a putative subunit of BCM. The amino acid sequence of the major cysteinyl peptide was determined. A cluster of hydrophobic residues was found in the COOH-terminal region. The amino acid sequences of two of the glycopeptides were found identical up to the 22nd residue. The small number of tryptic peptides, as well as the large amount of NH2- and COOH-terminal amino acids found in BCM indicate that this glycoprotein is made up of similar subunits with a molecular weight of about 22,000, one of the glycopeptides representing the NH2-terminal part, and one of the cysteinyl peptides, the COOH-terminal part. However, the existence of these subunits was not confirmed by ultracentrifugation of BCM in dithiothreitol and sodium dodecyl sulfate. BCM was polydisperse and had a mean molecular weight of 507,000.     

Characterization of intact and trypsin-digested biosynthetic human growth hormone by high-pressure liquid chromatography

The structural properties of purified human growth hormone (hGH) produced by Escherichia coli K-12 into which the hGH gene has been inserted have been fully characterized by high-pressure liquid chromatography of native hGH and tryptic digests of hGH. All of the tryptic peptides have been separated by high-pressure liquid chromatography and their sequence determined. Comparison of the primary structure with that of the purified pituitary-derived hGH has established the integrity of the biosynthetic hGH disulfide arrangement and amino acid sequence with the presence of an extra NH₂-terminal methionine.     

Synthesis of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes: "thymine replacement" analogs of thymidine for evaluation as anticancer and antiviral agents

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 two-carbon substituents [-C...C-X, X = I, Br; -C...CH; (E)-CH=CH-X, X = I, Br; -CH=CH2; -CH2CH3; -CH(N3) CH2Br], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. The 5-substituted (E)-CH=CH-I and -CH2CH3 compounds exhibited negligible cytotoxicity in a MTT assay (CC50 = 10(-3) to 10(-4)M range), relative to thymidine (CC50 = 10(-3) to 10(-5)M range), against a variety of cancer cell lines. In contrast, the C-5 substituted -C...C-I and -CH(N3)CH2Br compounds were more cytotoxic (CC50 = 10(-5) to 10(-6)M range). The -C...C-I and -CH2CH3 compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B) and HSV-1 TK+ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK+). This observation indicates that expression of the viral TK enzyme did not provide a gene therapeutic effect. The parent group of 5-substituted compounds, that were evaluated using a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV), and human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural C-aryl nucleoside mimics are inactive and/or weakly active antiviral agents.     

Variability of leaf photosynthetic characteristics in rice and its relationship with resistance to water stress under different nitrogen nutrition regimes

The negative effects of water stress on rice can be alleviated by NH₄⁺ nutrition. However, the effects of mixed nitrogen (N) nutrition (NO₃^? + NH₄⁺) on resistance to water stress are still not well known. To investigate the response of rice growth to water stress and its relationship with photosynthetic characteristics, a hydroponic experiment supplying different N forms was conducted. Compared with NO₃^? nutrition, mixed‐N and NH₄⁺ nutrition greatly alleviated the reduction of leaf area, chlorophyll content, and photosynthesis under water stress, whilst subsequently maintaining higher biomass. In contrast, water stress inhibited the root‐shoot ratios in NH₄⁺‐ and mixed‐N‐supplied plants, indicating reduced root growth and higher photosynthate availability to shoots. The following key observations were made: (1) a similar stomatal limitation and low proportion of activated Rubisco were observed among the three different N nutrition regimes; (2) increased mesophyll conductance in NH₄⁺‐ and mixed‐N‐supplied plants simultaneously stimulated leaf photosynthesis and improved the water use efficiency and (3), the maximum carboxylation rate and actual photochemical efficiency of photosystem II in NH₄⁺‐ and mixed‐N‐supplied plants were significantly higher than that in NO₃^?‐supplied plants, thus resulting in higher photochemical efficiency under water stress. In conclusion, mixed‐N and NH₄⁺ nutrition may be used to develop strategies for improved water stress resistance and stimulated biomass production under conditions of osmotic stress and possibly drought.     

Intracellular proteolytic processing of the heavy chain of rat pre-alpha-inhibitor. The COOH-terminal propeptide is required for coupling to bikunin

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides named bikunin and heavy chain 3 (H3). Both polypeptides are synthesized in hepatocytes and while passing through the Golgi complex, bikunin, which carries a chondroitin sulfate chain, becomes covalently linked to the COOH-terminal amino acid residue of H3 via its polysaccharide. Immediately prior to this reaction, a COOH-terminal propeptide of 33 kDa is cleaved off from the heavy chain. Using COS-1 cells transfected with rat H3, we found that in the absence of bikunin, the cleaved propeptide remained bound to the heavy chain and that H3 lacking the propeptide sequence did not become linked to coexpressed bikunin. Sequencing of H3 secreted from COS-1 cells showed that part of the molecules had a 12-amino acid residue long NH2-terminal propeptide. Cleavage of this propeptide, which occurred in the endoplasmic reticulum, was found to require basic amino acid residues at P1, P2, and P6 suggesting that it is mediated by a Golgi enzyme in transit. Deletion of the NH2-terminal propeptide or blocking of its release affected neither transport nor coupling of the heavy chain to bikunin.     

Distinct structural elements that direct solution aggregation and membrane assembly in the channel-forming peptide M2GlyR

Restoration of chloride conductance via the introduction of an anion selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis (CF). Delivery of these peptide sequences to airway cells from an aqueous environment in the absence of organic solvents is paramount. New highly soluble COOH- and NH(2)-terminal truncated peptides, derived from the second transmembrane segment of the glycine receptor alpha-subunit (M2GlyR), were generated, with decreasing numbers of amino acid residues. NH(2)-terminal lysyl-adducted truncated peptides with lengths of 22, 25, and 27 amino acid residues are equally able to stimulate short circuit current (I(SC)). Peptides with as few as 16 amino acid residues are able to stimulate I(SC), although to a lesser degree. In contrast, COOH-terminal truncated peptides show greatly reduced induced I(SC) values for all peptides fewer than 27 residues in length and show no measurable activity for peptides fewer than 21 residues in length. CD spectra for both the NH(2)- and COOH-truncated peptides have random structure in aqueous solution, and those sequences that stimulated the highest maximal I(SC) are predominantly helical in 40% trifluoroethanol. Peptides with a decreased propensity to form helical structures in TFE also failed to stimulate I(SC). Palindromic peptide sequences based on both the NH(2)- and COOH-terminal halves of M2GlyR were synthesized to test roles of the COOH- and NH(2)-terminal halves of the molecule in solution aggregation and channel forming ability. On the basis of the study presented here, there are distinct, nonoverlapping regions of the M2GlyR sequence that define solution aggregation and membrane channel assembly. Peptides that eliminate solution aggregation with complete retention of channel forming activity were generated.     

Susceptibility of neuroactive peptides to aminopeptidase digestion is related to molecular size.

Peptide fragments derived from the NH₂-terminus of corticotropin were found to exhibit widely differing degrees of stability to degradation by aminopeptidase M. Corticotropin itself was 135 times more stable than its NH₂-terminal pentapeptide, and similar differences in stability were observed with peptides derived from the B-chain of bovine insulin. Enkephalin linked covalently to the A-chain of bovine insulin was at least 100 times more stable than the pentapeptide. The results demonstrate that the molecular size of a peptide is one factor that determines its NH₂-terminal stability.