Trypsin condensation

The following are topics that may be especially valuable to the student and which might be read initially in Chapter 12, lysozyme (Section B,5), chymo-trypsin (Section C,l), kinases (Section D,9), multiple displacement, reactions (Section G) in Chapter 13, imines (Section A,2), addition to C=C bonds (Section A, 4,5), beta cleavage and condensation (Section C) in Chapter 14, thiamin diphosphate (Section D), pyridoxal phosphate (Section E) in Chapter 15, NAD (Section A). 

Fluoromethyl ketones are one of the most widely used classes of peptidyl a-fluoroalkyl ketones, second only to trifluoromethyl ketones. Peptidyl fluoromethyl ketones are very effective as irreversible inhibitors of cysteine proteases the first reported use of a fluoromethyl ketone compound was the use of Z-Phe-Ala-CH2F as an irreversible inhibitor of cathepsin BJ2,31 Today, many lysine and arginine derivatives have been synthesized as potential inhibitors for trypsin and trypsin-like enzymesJ3 There are four basic methods for the synthesis of peptide fluoromethyl ketones (1) the reaction of HF with peptide diazomethyl ketones (Section 15.1.4.1.1), (2) a halogen-exchange reaction with a chloro-, bromo-, or iodomethyl ketone (Section 15.1.4.1.2), (3) a Henry nitro-aldol condensation reaction (Section 15.1.4.1.3), and (4) a modified Dakin-West acylation reaction (Section 15.1.4.1.4). 

Cytochrome synthesis was examined in the fat body of adult male B. discoidalis by measuring the synthesis of cytochrome hemes. Heme is synthesized from the condensation of succinate and glycine by aminolevulinic acid synthase to produce aminolevulinic acid (ALA), a specific heme precursor. A developmental pattern exists for the incorporation of [i CjALA into cytohemes of fat body mitochondria with a peak of synthesis between days 4 and 6 of adult age (60). CC ablation eliminates this peak of synthesis for cytohemes a and b CC extract injections return the synthesis of cytohemes a+b to normal levels in CC-ablated cockroaches but have no effects on the synthesis of the c-type hemes for cytochromes c and Cj, The synthesis of cytohemes a+b in response to CC extracts requires a latent period of 24-48 hr to obtain a maximum response and is dose-dependent over a range of 0.01 to 0.08 CC pair (61). The active factor in CC extracts is sensitive to chymotrypsin but not to trypsin. This "cytochromogenic hormone (CGH) is secreted on days 2-3 of adult age in males (62). Since maximal synthesis of cytohemes a+b occurs on day 4, CGH secretion on days 2-3 agrees with the earlier observation that CGH requires about 48 hr to produce its response (61). 

One of the major problems in nutritional exploitation of tree leaves is the presence of antinutritional and toxic factors (6). The leaves of M. oleifera contain about 1.4% tannins, whereas condensed tannins are not present. Total phenols in leaves ranges from 2.7 - 3.4% (6,100). The M oleifera leaves contain 5.0% saponins as diosgenin equivalent with phytate contents in the leaves of 3. %(6, 100). Activity of trypsin inhibitors and lectins is not detected in the M. oleifera leaves. Other antinntritional factors present in M. oleifera leaves are flatus factors (sucrose + raffinose + stachyose) at 5.6% (107). Nitrate (0.5 tmnol per 100 g) and oxalate (4.1%) are also present inM oleifera leaves (100). The presence of these antinutritional factors in leaves of M. oleifera decreases the bioavailability of other nntrients. However, extraction nsing 80% ethanol decreases the contents of some of these antinntritional factors (6). 

If then, in the reconstruction of albuminoids enzymes really play a part, it cannot be the digestive enzymes such as pepsin and trypsin, but rather erepsin and the amidases, which apparently must have condensing properties like the preceding, and be, in addition, capable of reacting on the final products of hydrolysis. However, we do not possess at the present time data on the synthetic r61e of erepsin and the amidases. 

The well known specificity of proteinases implies the use of specific amino acids (amides, esters) as acyl donors and—seldom—specific amino acid (derivatives) as acceptors in enzymatic peptide bond formation, since the same structural features of RCONHR that influence the rate of hydrolytic cleavage are also involved in the synthesis. Accordingly trypsin is well suited to the formation of a new -Arg-X or -Lys-X bond. As an example the transformation of the -Lys-AlaOH terminus of the B-chain of porcine insulin into -LysThrOBu of human insuhn may be mentioned. C-terminal Ala was removed by means of car-boxypeptidase A, trypsin-catalyzed condensation of the des-alanine peptide with threonine tert. butylester gave 73% of the ester of human insulin [33] (see also... 

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