International Hydrolyzed Protein

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Peptide hydrolases (peptidases or proteases, i.e., enzymes hydrolyzing peptide bonds in peptides and proteins, see Chapt. 2) have received particular attention among hydrolases. As already described in Chapt. 2, peptidases are divided into exopeptidases (EC 3.4.11 -19), which cleave one or a few amino acids from the N- or C-terminus, and endopeptidas-es (proteinases, EC 3.4.21-99), which act internally in polypeptide chains [2], The presentation of enzymatic mechanisms of hydrolysis in the following sections will begin with peptidases and continue with other hydrolases such as esterases. 

The Py-unit of the G protein stimulates a kinase (PARK, not shown), which phosphorylates the receptor. This reduces its af nity for the hormone and leads to binding of the blocking protein arrestin. The internal GTPase activity of the a-subunit hydrolyzes the bound GTP to GDP within a period of seconds to minutes, and thereby terminates the action of the G protein on the adenylate cyclase (3). 

EF-G seems to be the motor protein that drives translocation in the 30S subunit. When it is not attached to a ribosome, the EF-G GTP complex is very stable, but in its functioning location GTP is hydrolyzed rapidly. This occurs prior to translocation398 3983 and presumably causes an internal alteration in the ribosome that energizes it for the translocation step. 

In all cases the cells also utilize cAMP as an internal second messenger. For D. discoideum the components of the chemotactic-aggregation system include a 41-kDa cAMP receptor on the outside, adenylate cyclase, an extracellular diesterase that specifically hydrolyzes the cAMP to AMP, and a diesterase inhibitor protein.35 227-230 The inhibitor keeps the phosphodiesterase largely inactive initially, but when cAMP concentrations build up synthesis of the inhibitor is repressed and the cAMP is hydrolyzed, a necessary condition for retaining sensitivity of the receptors for the arriving pulses of cAMP. 

Peptidases are often classified as either exopeptidases or endopeptidases, depending on the positional specificity of the bonds they hydrolyze. Exopeptidases act at peptide bonds located at either the N or C terminus of the protein. Those acting at the C terminus are referred to as carboxypeptidases, those acting at the N terminus as aminopeptidases. Endopeptidases, on the other hand, act at peptide bonds internal to the polypeptide chain. 

VLDLs are synthesized in the liver and transport triacylglycerols, cholesterol and phospholipids to other tissues, where lipoprotein lipase hydrolyzes the triacylglycerols and releases the fatty acids for uptake. The VLDL remnants are transformed first to IDLs and then to LDLs as all of their apoproteins other than apoB-100 are removed and their cholesterol esterified. The LDLs bind to the LDL receptor protein on the surface of target cells and are internalized by receptor-mediated endocytosis. The cholesterol, which is released from the lipoproteins by the action of lysosomal lipases, is either incorporated into the cell membrane or re-esterified for storage. High levels of intracellular cholesterol decrease the synthesis of the LDL receptor, reducing the rate of uptake of cholesterol, and inhibit HMG CoA reductase, preventing the cellular synthesis of cholesterol. 

Cholesteryl esters that are internalized via the LDL receptor are hydrolyzed to produce cholesterol and an acyl chain. Cholesterol, in (urn, activates the enzyme acyl-CoA cholesterol acyl-transferase (ACAT) which re-esterifies cholesterol. In an apparently futile cycle, the cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase. The cholesterol moiety has several fates it may leave the cell and bind to an acceptor such as high-density lipoprotein (HDL), it may be converted to steroid hormones, or it may be reesterified by ACAT. When the cellular cholesterol concentration falls, the activity of HMG-CoA reductase is increased, as is the number of LDL receptors, which results in an increase of cellular cholesterol, due both to de novo synthesis and to the uptake of cholesterol-rich lipoproteins in the circulation. An increase in cellular cholesterol results in the rapid decline in the mRNA levels for both HMG-CoA reductase and the LDL receptor. This coordinated regulation is brought about by the presence of an eight nucleotide sequence on the genes which code for both proteins this is termed the sterol regulatory element-1. 

Katunuma and coworkers (1971) described a protease in the rat that hydrolyzes the apoenzymes of a number of pyridoxal phosphate-dependent enzymes it has no effect on other proteins or the holoenzymes. Presumably, it attacks the conserved amino acid sequence around the active lysine residue to which the internal Schiff base is formed. The activity ofthe enzyme is increased some 10- to 20-fold in vitamin Be deficiency, suggesting that its function is to degrade those enzymes that lose their coenzyme more readily, and so make more pyridoxal phosphate available for use by other enzymes. There is also evidence that some pyridoxal phosphate-dependent apoenzymes are modified to become incapable of activation by pyridoxal phosphate, although retaining immunological cross-reactivity with the normal form of the enzyme in vitamin Be deficiency (Nagata and Okada, 1985). 

Most dietary proteins are known not to be absorbed in humans as intact forms. Instead, they are usually broken down into amino acids or di- and tripeptides first in the GI tract. The stomach secretes pepsinogen, which is converted to the active protease pepsin by the action of acid. Pepsins, which are most active at pH 2-3, hydrolyze partially digested dietary proteins. The partially digested dietary proteins are further broken down by proteolytic enzymes (peptidases) produced by the pancreas and secreted in the duodenum of the small intestine. The peptidases that break the internal peptide linkages are known as endopeptidases, whereas those that attack the terminal, or end, groups of amino acids are called exopeptidases. 

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