Biomedica biochimica acta最新文献

D-Xaa6-GnRH analogs (Xaa: Ala, Nal1), Phe, Ser(tBu), Trp) were prepared by chymotrypsin catalyzed 3 + 7 fragment coupling synthesis with conversion rates of the amino component in the range from 90.3 to 97.4%. For D-Phe6-GnRH the method was scaled up to production level.

Synthesis of LHRH N-terminal pentapeptide Glp1-His2-Trp3-Ser4-Tyr5-OMe (6) involving 3 + 2 enzymatic coupling has been developed. Synthetic strategy features the formation of one peptide bond (Glp-His) by chemical coupling and three peptide bonds by means of papain (Trp-Ser, Ser-Tyr) or alpha-chymotrypsin (His-Trp). High efficiency of this six-step synthesis is demonstrated by 44% overall yield. Its advantages are the use of inexpensive enzymes, simple isolation of intermediates and final pentapeptide, and easy recovery of substrates.

Thermolysin is an effective peptide synthetase in mixtures of buffer and water miscible solvents MeOH, EtOH and PrOH. Although the solvents are inhibitory, primarily through effects on kcat, synthetic reactions are much less affected than peptide hydrolysis. This may be of value in prevention of unwanted proteolytic cleavages, for example in protein semisynthesis.

Peptide synthesis with chymotrypsin in organic solvents was investigated and the apparent partition constants have been measured. We find that the Papp values of the most amino acids and peptide derivatives are drastically changed and the stereo- and regiospecificity in acetonitrile/water mixture is reduced.

In the experiments presented here 22 monoclonal antibodies (MoAbs) were produced which reacted with the tumor marker carcinoembryonic antigen (CEA). Eleven of the MoAbs reacted neither with peripheral blood granulocytes nor with purified spleen NCA-60 kDa and were therefore regarded as "CEA-specific". Only three antibodies of this group reacted exclusively with CEA-180 kDa. Eight MoAbs reacted with CEA-180 kDa and with CEA-like substances of lower molecular mass (of 160 kDa and/or 120 kDa) present in colon carcinoma cells as determined by immunoblotting. These molecules seem to be different from the classical non-specific cross-reacting antigens (NCAs) present in peripheral blood granulocytes. In contrast to that, the other 11 anti-CEA MoAbs recognized in addition to CEA-180 kDa also NCAs on granulocytes. Six of them were reactive with a purified spleen NCA-60 kDa preparation. These MoAbs bound also to reduced and alkylated CEA-180 kDa (CEA r/a), i.e. they recognize sequential epitopes. All 22 MoAbs reacted with CEA expressed in different human tumor cell lines as determined by immunocytological analysis. But six of them did not bind to the surface of these cells when tested in a radioimmuno-binding assay. It was concluded that the epitope(s) recognized by these antibodies are involved in cell membrane anchoring of the CEA-molecules.

Tissue-type plasminogen activator (t-PA) binds to heparin with an association constant of 2.4 x 10(4) l/mol at pH 7.4. The binding increases at lower pH-values and reaches maximal values below pH 6.0. Sodium chloride above 0.5 mol/l abolishes t-PA binding to heparin at neutral pH. t-PA binds to lysine maximally at pH 6 to pH 9. At neutral pH the association constant is 1.8 x 10(5) l/mol. Sodium chloride concentrations of 1 mol/l reduce interaction of the enzyme to lysine by about 60% at pH 7.4. Binding of t-PA to fibrin and to partially plasmin-degraded fibrin takes place maximally at pH 6 to 9. The interaction of t-PA with plasmin-degraded fibrin is less sensitive to addition of lysine than the interaction of t-PA and fibrin. Sodium chloride concentrations of 1 mol/l reduce the interaction of t-PA to fibrin by about 60% at pH 7.4.

A stimulated autotransplantation of the rectus femoris muscle in the rabbit was performed by clamping of the blood vessels for 2 h and cutting and suturing of the nerve. Four months after this operation, isometric contractions of the reinnervated muscle were recorded. Comparing controls and postoperation cases, the recovered strength measured about 65%. Due to operation stress, the epinephrine content in plasma was about 420 pg/ml; in plasma of the ischemic muscle it was enhanced to more than 600 pg/ml. During or immediately after ischemia, conjugated epinephrine is assumed to be converted to the free hormone. In systemic blood additionally about 200 pg/ml epinephrine were identified as glucuronides. The activity of adenylate cyclase increased, while the acetylcholine esterase decreased to one-half after nerve cutting.

A cDNA clone, with an insert of 3068 bp, encoding the N-terminus of human tripeptidyl peptidase II (TPP II) has been isolated and sequenced. Together with previous data (GenBank accession no J05299) a total of 4634 bp of the nucleotide sequence for TPP II can now be assembled. There is an in-frame methionine at position 8, which probably represents the starting point of translation, indicating that the complete coding sequence for TPP II now has been determined.

Bilateral symmetric lesions of a small strip in the dorsal pontine reticular formation between the locus coeruleus and the laterodorsal tegmental nucleus caused a severe disturbance of kidney and urinary bladder function beginning with haematuria, micturition paralysis and incontinence on the second postoperative day. Thirteen rats with these lesions were killed on the sixth postoperative day, dissectioned and their kidneys were pathomorphologically investigated. Their urinary bladders were extremely expanded and filled with bloody urine. All kidneys showed a picture of hydronephrosis and were enlarged from 15 mm preoperatively to 30 mm in their longitudinal axis and from 9 to 15 mm in their transverse axis. In seven further rats with partial or asymmetric lesions of this pontine micturition area the micturition cessation and the haematuria disappeared between the 4th and 8th postoperative days: kidney enlargement and hydronephrosis were still found on the 20th postoperative day.

Lysosomes and autolysosomes were isolated from the livers of dextran-treated and leupeptin-injected rats, respectively. The contents of sequestered cytoplasmic proteins were much higher in autolysosomes, and more than 50% of them were found in the sediment. Isolated dextran-lysosomes showed high proteolytic activity toward sequestered cytoplasmic enzymes following incubation at pH 5. E-64 caused a complete inhibition of their degradation. Leupeptin treatment caused enrichment of ubiquitin protein conjugates of high molecular mass in the sediment of autolysosomes, which is comparable to enrichment of carbamyl-phosphate synthetase as a marker of bulk cytoplasmic sequestration in autolysosomes. The results suggest that protein ubiquitination may not be a signal for sequestration of proteins into the autophagic pathway.