氨基酸胜肽.Amino-Peptide

Amino Acids and their Functions:

According to David Spindel, "amino acids are organic molecules that form the basic constituents of protein. " They are the "building blocks" of the body. They build and repair various body parts and aid certain body functions. There are twenty-two amino acids. Eight are essential and fourteen are non-essential. The body does not manufacture the essential acids and does manufacture the non-essential acids. The body contains a free amino acid pool that contains tissues and bodily fluids. Amino acids enter this pool by three ways. Amino acids enter during digestion of foods containing protein, when body protein decomposes, and when carbon sources and NH3 synthesize the non-essentials. When protein intake is insufficient, there are not enough amino acids entering the pool to compensate for the lost ones. This affects the muscle size and strength . Amino acids aid the body in many ways, and are definitely needed by the body to function properly. Supplements are needed only when the body does not get the required amounts. The purposes of amino acid supplements are to replenish the lacking acid supply and in most sports uses, to stimulate lean mass growth without sacrificing present muscle mass.

 

Major histocompatibility complex (MHC) class II molecules bind antigenic peptides for display to T lymphocytes. Although the enzymes involved remain to be identified, it is commonly believed that class II associated peptides are released from intact antigens through a series of proteolytic steps carried out inside antigen presenting cells. We have examined the effect of amino acid substitutions on proteolytic processing of the model antigen hen-egg lysozyme (HEL). Altered HEL molecules, engineered by site-directed mutagenesis of a HEL cDNA, were expressed as separate stable transfectants in a B cell lymphoma line. Each transfectant processed a different mutant HEL protein for presentation on MHC class II. We purified the resulting class II-associated peptides and analyzed them by mass spectrometry. Our results strongly support the hypothesis that antigen processing continues after peptide binding to the MHC class II molecule and are most consistent with a scenario in which long peptides first bind to MHC class II and are then trimmed by exopeptidase.

 

INTRODUCTION:  

It has been proposed that major histocompatibility complex (MHC) class II binding protects peptides from degradation during intracellular processing (1). This idea has been supported by the ability of affinity-purified class II molecules to protect bound peptides from protease digestion in vitro . However, it is still unclear whether this protection occurs in vivo.

 

When bound, only the core of a peptide (about  amino acids) is in contact with the class II molecule, leaving the peptide ends free to extend beyond the binding site ). Two lines of evidence suggest that these ends are available for trimming by exopeptidase enzymes. First, naturally processed peptides vary greatly in length, with most class II-associated peptides reported to span between 13 and 17 amino acids . Often naturally processed peptides belong to nested sets that share a common core sequence but differ at both the amino and carboxy termini. This length heterogeneity around a common core sequence suggests trimming of the peptide ends by exopeptidases.

 

Second, pool sequencing has revealed that proline residues occur with high frequency near the ends of naturally processed peptides, with proline especially common as the penultimate amino-terminal residue . Proline is known to block the ability of several exopeptidase enzymes to remove amino- and carboxyl-terminal amino acids from peptides  (due to the unusual chemical structure of the peptide bond formed by the imino group). The high frequency of proline residues near the ends of naturally processed peptides is consistent with the idea that these ends result from trimming by exopeptidase enzymes.

 

In this study, we examine the sequence specificity of antigen processing to obtain direct evidence for the amino-terminal trimming of naturally processed peptides.

 

MATERIALS AND METHODS:  

 

Expression Vector Assembly.

To create a membrane hen-egg lysozyme (mHEL) fusion construct, the entire gene for HEL was joined in-frame to a portion of the MHC class I Ld gene (see Fig. ). A -kb KpnI fragment from the plasmid pLd containing part of the Ld genomic sequence was moved to Bluescript KS(). A second NaeI site was created on this plasmid in the Ld domain by oligonucleotide-directed site mutagenesis [changing nucleotides as numbered by Linsk et al. (13)]. The resulting 3-kb NaeI fragment containing the end of the Ld gene was purified and inserted at the naturally occurring NaeI site found in the penultimate amino acid codon of a HEL cDNA, also in Bluescript KS(). The codons for the final  amino acids of the HEL protein, lost after cleavage with NaeI, are restored in the fusion gene. Further site-directed mutagenesis was performed on this plasmid to generate a set of different mutant mHEL genes. For the final step in the construction, NotI-XhoI fragments containing the different HEL/Ld fusion genes were moved into the NotI-SalI sites of the expression vector pCEP4. 

 

 

Fig. . Schematic representation of the mHEL expression vector. A cDNA encoding the entire HEL sequence (129 amino acids) was joined in-frame to a genomic portion of the MHC class I H-2Ld gene encoding a connecting stalk (8 amino acids), a transmembrane region (TM;  amino acids), and a complete cytoplasmic domain (CY; 25 amino acids). Transcription of the fusion gene was driven by the promoter region of the human cytomegalovirus (CMV) major immediate early gene. The construct extends through the genomic termination of transcription and polyadenylylation sequences used by Ld.

Structure Chart of Amino Acids

 

 

 

Purpose and Rationale:

Many sources on the Internet encourage people who want to lower their caloric intake and increase their protein intake to use amino acids. By using amino acids, individuals can achieve better health by meeting the bodies needs. Amino acids achieve their goals in different ways. Every product on the market has specific instructions for usage. Essential Fitness recommends taking amino acid supplements with meals. They claim that food acts as a "buffering agent" for the acids, that food without proper acids can be enhanced with supplements, and that as more insulin is released during meals, the acids will be better accepted by the body. Other products such as Aminolyze recommend taking "one tablespoonful with water on an empty stomach at bedtime." Regardless of the usage instructions, all amino acid supplements add amino acids to the body to be broken down for use by the body. Some of these uses include "assisting in transporting long chain triglycerides…stimulating the pituitary to secrete growth hormone…supplying the body with nitrogen…and much, much, more." Many of these functions add to lean muscle mass, which is the primary purpose of many supplements available.

 

 

　

This structural information, as well as other published data (1, 4, 37–45), were used to align and predict the anchor residues of pMCC and pPCC(T102S), as well as other peptides that are the dominant IEk epitopes in foreign protein antigens, or major self-peptides isolated from IEk naturally present on the surface of B cells, and thymic epithelium in unimmunized mice (Fig. 2) . This alignment predicts an aliphatic amino acid at the p1 position. The p4 pocket is predicted to be more forgiving, preferring hydrophobic amino acids such as Phe and Leu, but also allowing other amino acids. The p6 pocket appears to have a preference for amino acids that can participate in the hydrogen bonding network underlying the peptide (Glu, Gln, and Asn), but small side chains such as the Ala in pHSP are also allowed. The p9 pocket is predicted almost invariably to be Lys.

 

The amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide. The substitution may be a conserved substitution. A"conserved substitution"is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide retains its spacial conformation but has altered biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln ; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 common amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains.

 

It is known that variant polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve biological activity. For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide that result in increased bioactivity. Alternatively, amino acid substitutions in certain polypeptides may be used to provide residues that may then be linked to other molecules to provide peptide-molecule conjugates that retain sufficient properties of the starting polypeptide to be useful for other purposes.

 

The variant cPepTl protein comprises at least seven amino acid residues, preferably about 20 to about 700 residues, and more preferably about 50 to about 700 residues, wherein the variant cPepTl protein has at least , preferably at least about , and more preferably at least about  but less than , contiguous amino acid sequence homology or identity to the amino acid sequence of a corresponding native cPepTI protein.

 

The amino acid sequence of the variant cPepTl protein corresponds essentially to the native cPepTl protein amino acid sequence. As used herein "correspond essentially to"refers to a polypeptide sequence that will elicit an absorption value substantially the same as the absorption stimulated by native cPepTl protein. Such absorption may be at least  of the level generated by native cPepTl protein, and may even be at least  of the level generated by native cPepTl protein.

 

A variant of the invention may include amino acid residues not present in the corresponding native cPepTl protein, or may include deletions relative to the corresponding native cPepTl protein. A variant may also be a truncated "fragment"as compared to the corresponding native cPepTl protein, i. e., only a portion of a full-length protein. cPepTl protein variants also include peptides having at least one D-amino acid.

 

Cyclic Peptides:

Cyclic peptide analogues of biologically active peptides are included in the

sections dealing with individual peptides (Section 4). Sequences, synthetic

routes and biological activities of other cyclic peptides isolated from natural

sources are presented here. Synthetic routes to cyclic peptides and depsipeptides on various solid supports either by attaching the amino acid

through the C-terminal carboxyl group, a-nitrogen atom or the side chain

functional groups are reported and work on safety catch linkers and monitoring

techniques has been published.For example, synthesis of a cyclic

peptide containing a R-3-hydroxy-13-methyltetradecanoic acid residue, cyclo-

(Gln-Leu-d-Leu-Val-Asp-d-Leu-Ile-O-CH((CH2)9-CHMe2)-CH2CO), was achieved by using a cyclisation-cleavage method with oxime resin.38 A 4-alkoxybenzyl- derived linker that anchors the C-terminal amino acid to the resin through the a-nitrogen atom was used to synthesise the cytotoxicheptapeptide,

  

Free and peptide amino acid net flux across the rumen and the mesentericand portal-drained viscera of sheep:

D. Re´mond1, L. Bernard, and C. Poncet

Unite´ de Recherches sur les Herbivores, Institut National de la Recherche Agronomique de Clermont-Ferrand,

63 122 St Gene`s-Champanelle, France

 

ABSTRACT: This experiment was conducted to determine:

the significance of the peptide amino acid (PAA) contribution to amino acid (AA) net flux in the portal vein and to evaluate the capacity for peptide absorption in the different segments of the gastrointestinal tract of ruminants. Four sheep  were fitted with catheters and blood flow probes, allowing AA

net flux measurements across the portal- (PDV) and mesenteric (MDV)-drained viscera and the rumen. Sheep were fed at maintenance a diet containing hay and extruded peas . Peptide absorption was investigated by a dose infusion of a mixture of peptides (casein hydrolysate, Pro-Phe, β-Ala-His, Gly-Gly) into the rumen. Control and postinjection net fluxes of plasma free amino acids (FAA) and PAA were determined. The concentration of plasma PAA was determined by quantification of amino acids before and after acid hydrolysis of samples first submitted to chemical

deproteinization and ultrafiltration (3-kDa cut-off filter). During the control period a significant net release  PAAwas observed across the PDV, which Key Words: Absorption, Amino Acids, Peptides, Sheep American Society of Animal Science. All rights reserved. J. Anim. Sci.

 

Amino Acids and Peptides----

Outline:

• Acid-base equilibrium
• The Henderson-Hasselbach Equation
• Amino acid structures
• Chirality
• Amino acids: acid/base chemistry
• Side-chain reactivity
• Peptides and proteins
• Side-chain reactivity in peptides and proteins
• Disulfides

Proteins are made up of amino acids, and free amino acids and oligomers of amino acids (peptides) play important roles in cells. We discuss amino acids and peptides in terms of their structures and functions.

Acid-base equilibrium

Almost all biochemical reactions take place in aqueous solution, for which there is a nonzero concentration of protons (hydrogen ions) and hydroxide ions present. By definition the pH of a solution is the negative base-10 logarithm of the hydrogen ion concentration; similarly, pOH is the negative logarithm of the hydroxide ion concentration. The product of the hydrogen ion concentration and the hydroxide ion concentration is almost precisely .

Neutral pH refers to the condition under which pH = pOH; clearly pH  under those conditions. Pure water is at pH=pOH, but pH can occur even with externally-introduced ions present.

The Henderson-Hasselbach equation:

A fundamental equation regarding ions in aqueous solution relates the acid-base equilibrium to the the equilibrium constant for the ionization of a solute.

This is the Henderson-Hasselbach equation. With it we can determine the [base]/[acid] for a solute. Several of the problems in chapter 3 depend on this equation.

Amino acid structures:

An amino acid is any molecule that contains both a carboxyl group and an amine group. The amino acids that serve as building blocks of proteins are alpha-amino acids, i.e. molecules in which the amine group and the carboxyl group are separated by one intervening saturated carbon atom:

H3N+-CHR-COO-

There are amino acids of relevance to biochemistry that are not alpha-amino acids, such as beta alanine:

H3N+-CH2-CH2-COO-

but we will concentrate on the alpha-amino acids today.

Note that we depict amino acids as zwitterions, i.e. molecules that contain both a positive charge and a negative charge. At extremes of the pH range, an amino acid will not be a zwitterion. At very low pH the carboxyl group will become protonated:

H3N+-CHR-COOH

At very high pH the amine group loses a proton and becomes uncharged:

H2N-CHR-COO-

These acid-base equilibrium phenomena are instances of the reactivity of free amino acids.

Among the alpha amino acids the only variation possible from one to another is in the identity of the R group. The simplest R consists of a hydrogen atom: in that instance, the amino acid is glycine. The next simplest R is a methyl (CH3) group; this is alanine. they range from the simplest ones (glycine and alanine) up to tryptophan, which contains a fused double-ring system.

A special case, but nonetheless one of the amino acids coded for by the ribosomal protein synthetic system, is proline. It is not an amino acid at all: it is an imino acid, because the amine group is covalently bonded through three methylene groups to the alpha carbon. Thus the alpha carbon and the amine nitrogen contribute to a five-membered ring. This structure is more restricted rotationally than the ordinary amino acids. We will see the implications of that restriction in proline's role in proteins.

Chirality:

Every alpha-amino acid except glycine is a chiral molecule; that is, it contains at least one carbon atom with four unequivalent substituents, so that the molecule is not superimposable upon its mirror image. Glycine is nonchiral because two of the substituents on its alpha carbon are hydrogens. Two of the amino acids--isoleucine and threonine--have a second chiral center on the side chain.

The amino acids that make up proteins are all L-amino acids; that is, the substituents on the alpha carbon are arranged in a specific order that relates them to a reference molecule that rotates polarized light in the leftward direction. The mirror image of an L-amino acid is a D-amino acid; it is related to a reference molecule that rotates polarized light to the right. D-amino acids do play a role in a few biochemical systems, such as bacterial cell walls and some antibiotics, but they are not synthesized by the ribosomal apparatus. It is not accidental that bacteria incorporate these D-amino acids into their cell walls: most of the proteolytic enzymes (enzymes that cleave peptide bonds) that the hosts of these bacteria produce in order to destroy the bacteria act only against L-amino acids, so including D-amino acids in their cell walls confers a competitive advantage to these bacteria.

To remember what the absolute configuration of an L-amino acid is, we use the mnemonic CORN. Envision the amino acid, arranged so that you are looking down the bond from the alpha hydrogen to the alpha carbon. Specifically, you are looking from the H to the C, and examining the order in which the other three substituents on the alpha carbon appear. One of those three will be the carbonyl carbon (CO); the next, as you go around clockwise, will be the side chain (R); the last will be the nitrogen (N). Thus, CORN. For this mnemonic to be useful you must remember to start by looking down the H-C bond, and you have to remember to traverse the other three substituents clockwise. A D-amino acid would be NROC.

Amino acids: acid/base chemistry:

The most obvious chemistry in which free amino acids can participate involves acid-base equilibrium at the main-chain carboxyl and amine groups:
H3N+-CHR-COO- + OH-<-> H2N-CHR-COO- + H2O
and
H3N+-CHR-COO- + H+ <-> H3N+-CHR-COOH

The equilbrium in these reactions is far to the left at pH values close to neutral, but at low pH equilibrium in the second reaction will lie somewhat to the right, and at high pH equilibrium in the first reaction will lie somewhat to the right.

The pKa values for these reactions--the pH values at which the reactions depicted above have  products and  reactants--depend somewhat on what sidechain (R group) is present. Thus since the pKa for deprotonating the amine group in alanine is, an aqueous solution of alanine at pH  will be half in the protonated (H3N+) form and half in the deprotonated (H2N) form. The pKa for the amino group of threonine is lower--about . So at pH , more than half of the threonine in a solution will be deprotonated, whereas less than half the alanine will.

Every free amino acid has at least two pKa values: the one associated with protonation of the carboxylate and the one associated with deprotonation of the amine group. Amino acids in which the side-chain itself contains an ionizable group have a third pKa value--the one one associated with protonation or deprotonation at the side chain. The full collection of pKa values appears in the table below,

　

　

Side-chain reactivity:

From the table above it is clear that one of the ways in which amino acid side-chains can participate in chemical reactions is through acid-base interactions. But other kinds of chemistry occurs in side-chains as well. Sulfur atoms in cysteine and methionine can become oxidized to sulfates, sulfites, and related forms. The side-chain hydroxyl groups of serine and tyrosine can form covalent bonds to ligands, such as phosphate groups. Both of the nitrogen atoms in the imidazole side-chain of histidine can covalently bond to various ligands.

Not all side-chain reactivities involve formation of covalent bonds. Side-chain polar groups can form hydrogen bonds with other polar groups. "Salt bridges" between oppositely charged groups (e.g. the side-chain terminal amine group of lysine and the side-chain carboxylate of aspartate) are often found in proteins.

Peptides and proteins:

Peptides and proteins are, respectively, oligomers and polymers of amino acids. Most are heteropolymers, i.e. the individual building blocks are not all identical. Chemists can manufacture homopolymers of amino acids, in which all the building blocks are identical, but these do not play roles in real biological systems.

A dipeptide is produced, formally, by removing water from two amino acids:
H3N+-CHR-COO- + H3N+-CHR-COO- -> H3N+-CHR-CO-NH-CHR-COO- + H2O
The covalent bond between the carbonyl carbon in the center of this assembly and the amide nitrogen is called an amide bond or peptide bond. The C-N bond has some double bond character because of a resonance in which the carbonyl oxygen can take on a formal negative charge and the amide nitrogen can take on a formal positive charge. This partial double-bond character obliges the six atoms of the peptide group--the main-chain carbonyl carbon, the carbonyl nitrogen, both adjoining alpha carbons, and the hydrogen attached to the nitrogen-- to lie in a single plane, termed the peptide plane. Study fig. 4.6 to see how this works.

Building up a tri-, tetra-, oligo-, or polypeptide is accomplished, formally, in the same way as the creation of the dipeptide:
H3N+-CHR-CO-(NH-CHR-CO-)n NH-CHR-COO- + H3N+-CHR-COO- -> H3N+-CHR-CO-(NH-CHR-CO-)n+1 + H2O
The way this is usually accomplished in the cell is within the mechanisms of the ribosome, where the lengthening of the polypeptide chain is accomplished under careful enzymatic control. The ligation reaction, in which the chain is lengthened by one residue, is endergonic, and the energy required to drive it is obtained from hydrolysis, not of our familiar energy currency ATP, but rather of its cousin GTP:
GTP + n-length-peptide + amino acid -> GDP + Pi + (n+1)-length peptide

Ordinarily there is a free amine group at one end of the polymer and a free carboxylate at the opposite end:
H3N+-CHR-CO-(NH-CHR-CO-)n -NH-CHR-COO-
But occasionally cyclic peptides are formed, in which the chain bends around and a peptide bond is formed between the terminal amine group and the terminal carboxyl group, with the usual elimination of water. This type of cyclization is not carried out at the ribosome; it is carried out by a specific enzymatic synthesis.

Side-chain reactivity in peptides and proteins:

We have already mentioned that the side chains in peptides and proteins can undergo acid-base interactions. It is worth noting that in an intact protein, these are the only acid-base interactions that can occur, except for those involving the terminal amino and carboxyl groups. The amide nitrogens and carbonyl groups are fully engaged in peptide bonds in all the amino acids between the second and next-to-last amino acids in a protein and therefore cannot participate in reactions without rupturing the polypeptide chain.

Protein side chains do show other kinds of reactivity as well, in similar ways to those mentioned above in the contexts of free amino acids. The reactivities of side chains in intact proteins differ somewhat from the reactivities of side chains in free amino acids, in that the molecular environment in which the side chain finds itself is likely to be different from that of a free amino acid. Thus a zwitterionic free amino acid, even if it has a hydrophobic side chain, tends to end up in a fairly hydrophilic (water-loving) environment, so its reactivity will be characteristic of an aqueous species. By contrast, an amino acid with a hydrophobic side chain in an intact protein will usually be found in a hydrophobic environment, and its reactivity will be altered appropriately.

Disulfides:

Disulfides are covalent bonds between sulfur atoms. The amino acid cysteine can participate in disulfide bonds. The formation of a disulfide is an oxidation-reduction reaction:

R-SH + R'-SH + 1/2O2 --> R-S-S-R' + H2O

In this equation I have designated the oxidizing agent as dioxygen; other oxidizing agents can in fact operate to produce the disulfide.

The only one of the twenty ribosomally-encoded amino acids that can produce disulfide bonds is cysteine, for which

R = (NH3)+-CH(CH2)-COO-

where the connection to the sulfur is on the methylene group in the middle. When two cysteine residues are oxidized to produce the disulfide, the resulting species (R-S-S-R') is sometimes (especially in the older literature) known as a cystine moiety. Within a protein, a disulfide bond can be produced from a pair of cysteine residues that are far apart in amino acid sequence, as long as there is an energetically favorable way to bring the two cysteines spatially close to one another. Some proteins, especially those that operate in an oxidizing environment, contain one or more disulfides that are important to their stability.

The Effect:

The Amino-Peptide Complex

The Amino-Peptide Complex in the new line uses the latest pentapeptide technology combined with essential vitamins, B and E. Upon application, this unique Complex is absorbed into the skin where it boosts the skin's natural surface cell turnover process to reveal newer skin and enhances its appearance-without irritation.

According to Lauren Thaman Hodges, Associate Director, Global Skin Science, P&G, "The results of the NIH-supported study suggested that pentapeptides could offer important cosmetic benefits. Our scientists then validated these benefits and used our research to create a highly effective consumer anti-aging product.

Peptides are important for the skin’s natural healing process. Research on wound healing found that peptides, tiny chains of amino acids found in collagen, play a critical role in signalling skin cells to repair themselves.

Signal peptides are known for their roles in wound healing, endocrine function and nerve transmission. When a signal peptide (Pal-KTTKS) is applied to the skin, special cells start generating proteins necessary to produce new, healthy skin, according to a Procter & Gamble (P&G) press release.

The amino-peptide complex uses the latest pentapeptide (KTTKS) technology combined with essential vitamins B and E. Pentapeptide (KITTKS) is a strand of five amino acids that was discovered in skin healing research to regenerate damaged skin. Olay claims that it renews skin’s outer layer to help reduce the appearance of wrinkles and improve skin condition.

This breakthrough is an exclusive nighttime Amino-Peptide Complex. It works with a time-release effect, hydrating hour after hour to increase cell renewal within skin's surface.