Primary structure of CC-III, the glycosylated cysteine proteinase from the latex of Carica candamarcensis Hook.

The amino acid sequence of the cysteine proteinase CC-III from the latex of the subtropical species Carica candamarcensis Hook has been determined with the exception of seven residues (pos. 180-186). It was deduced from the sequence analysis of the whole chain and peptides obtained by tryptic, chymotryptic, peptic and thermolysinolytic hydrolysis. CC-III consists of 214 amino acid residues. Out of a total of eight cysteine residues, six are located at positions involved in the formation of the three disulfide bridges stabilizing the structure of papain related enzymes. CC-III from Carica candamarcensis is a glycoprotein with the carbohydrate moiety bound to asparagine at position 44. Out of 210 residues compared with the sequences of the four cysteine proteinases of Carica papaya L., CC-III shares 125 identical ones (59.5%) with papain, 142 (67.6%) with papaya proteinase IV, 146 (69.5%) with papaya proteinase III and 156 (74.3%) with chymopapain. All amino acid residues constituting the active site and subsite S2 in chymopapain are conserved in CC-III with the exception of the substitution Leu157--> Val in the latter. This fact as well as the highest degree of identity between CC-III and chymopapain point to a similar specificity of both enzymes and thus CC-III might be a suitable substitute for chymopapain as a chemonucleolytic agent.

Isolation and preliminary characterization of the cysteine-proteinases from the latex of Carica candamarcensis Hook.

The cysteine-proteinase chymopapain from Carica papaya L. is used for chemonucleolysis of damaged human intervertebral spinal discs. The purification of this enzyme is difficult. To overcome these problems, we were looking for a substitute among the cysteine-proteinases of Carica candamarcensis Hook. The latex from unripe fruits was collected in an aqueous solution of methylethanethiolsulfonate to prevent proteolytic activities. The soluble fraction of the lypophilized product provided four enzymatically active peaks (CC-I-CC-IV) during chromatography on CM-Sephadex C-50 in sodium acetate buffer, pH5.0. They could be further purified by rechromatography under similar conditions. The isolated enzymes have been characterized by PAGE, analysis of the Fourier transform infrared spectra, preliminary studies of their specificities as well as a comparison of the N-terminal amino-acid sequences up to position 43. CC-III proved to be glycosylated. CC-I and CC-III from Carica candamarcensis Hook are suggested to correspond to papain and chymopapain from Carica papaya L., respectively.

The thiol proteinases from the latex of Carica papaya L. IV. Proteolytic specificities of chymopapain and papaya proteinase omega determined by digestion of alpha-globin chains.

The proteolytic specificities of chymopapain and papaya proteinase omega were investigated by using the alpha-chains of manatee and mole haemoglobin, whose primary structures are known, as substrates. The resulting peptides from each enzymatic cleavage were isolated by gel filtration on Sephadex G-25, followed by reversed-phase HPLC of the separated peaks and, in some cases, further purified by preparative thin-layer electrophoresis. The purified peptides were then identified on the basis of their amino-acid composition. The proteolytic specificities of chymopapain and papaya proteinase omega, deduced from the experimental cleavage patterns, are compared to that of papain. As in the case of papain, the specificity-determining factor is the amino-acid residue of the substrate that will be bound in subsite S2 (the next but one from the scissible bond). Aromatic residues in this position, preferred by papain, are not important for chymopapain and papaya proteinase omega. Cleavages preferentially occur when S2 is occupied by leucine, valine or threonine. For chymopapain, proline in position S2 also causes cleavage.

The thiol proteinases from the latex of Carica papaya L. III. The primary structure of chymopapain.

The amino-acid sequence of chymopapain is presented. It was isolated from the latex of the fruits from the tropical species Carica papaya L. and is, besides papain and papaya proteinase omega, the third thiol proteinase from this source. The primary structure contains 218 amino-acid residues. It was deduced from sequence analysis of the native enzyme and of peptides obtained by tryptic, chymotryptic, peptic, thermolysinolytic and mild acidic hydrolysis. Out of a total of eight cysteine residues, six are involved in the formation of three disulfide bonds, the location of which has been established with the help of peptic and thermolysinolytic peptides and fragments, obtained by mild acidic hydrolysis. Chymopapain shares 126 identical amino-acid residues (58%) with papain and 141 (65%) with papaya proteinase omega, including the three disulfide bridges and the free cysteine in position 25, required for activity. Except some amino-acid residues in the substrate-binding site, all residues involved in the catalytic mechanism are conserved. The homology between papaya proteinases is discussed.

The influence of experimental conditions on the spectrin-hemoglobin interaction.

Human spectrin, when isolated, purified and stored in such conditions that preserve its tetrameric form, is able to associate with human hemoglobin as it is clearly shown by gel filtration. However, this hemoglobin-spectrin association does not seem to have a significant effect on hemoglobin oxygenation as indicated by equilibrium and rapid kinetics measurements.

Separation and characterization of four enzyme forms of beta-galactosidase from Saccharomyces lactis.

beta-Galactosidase from Saccharomyces lactis has been purified to serve as a model for the kinetic behavior of human lactase in adult lactase deficiency. Enzymes from both species are neutral and follow Michaelis-Menten kinetics. beta-Galactosidase of S. lactis is more readily available than the human lactase. An enzyme preparation from S. lactis (Maxilact 40,000), which is used commercially to hydrolyze lactose in milk, has been found to contain four isozymes of beta-galactosidase. Methods have been developed for the separation and purification of each of the four enzymes. The enzymes were found to differ in molecular mass, kinetic behavior, isoelectric point, response to pH, specific volume and sensitivity to metal ions. The four enzymes had apparent molecular masses of 630 kDa, 550 kDa, 41 kDa and 19 kDa. Their specificity constants (kcat/Km) were found to be 42.0, 355.2, 0.38 and 0.48 mM-1 s-1, respectively. The techniques of reiterated ultrafiltration used for the isolation of these isozymes may be applicable to other purification processes.

The thiol proteinases from the latex of Carica papaya L. I. Fractionation, purification and preliminary characterization.

Three thiol proteinases, namely papain, chymopapain and proteinase omega were purified to homogeneity from the latex of Carica papaya L. During the purification procedure, the thiol function of the cysteinyl residues were protected either as mixed disulfides with cysteamine or 2-thiopyridone or as S-sulphenylthiosulfate derivative or after blocking with p-chloromercuribenzoic acid. In marked contrast with earlier publications, chymopapain also was found to be a monothiol proteinase as papain and proteinase omega. The active sites of chymopapain and proteinase omega could not be distinguished from that of papain neither by the analysis of the pH dependence of kcat/Km nor by the examination of the pH dependence of the fluorescence emission spectra.

The thiol proteinases from the latex of Carica papaya L. II. The primary structure of proteinase omega.

The complete primary structure of the proteinase omega isolated from the latex of the Carica papaya fruits is given. The polypeptide chain contains 216 amino-acid residues, the alignment of which was deduced from sequence analyses of the native enzyme, the tryptic, chymotryptic, peptic and thermolysinolytic peptides and facilitated due to the considerable degree of homology with papain and actinidin. The location of the three disulfide bridges could be established with the help of peptic and thermolysinolytic fragments. Proteinase omega shares 148 identical amino-acid residues (68.5%) with papain and 108 ones (50%) with actinidin, including the three disulfide bridges and the free cysteine residue required for activity, as well as most of the other amino-acid residues involved in the catalytic mechanism and two thirds of the glycine residues which are of structural significance. The homology with other cysteine proteinases of different origin is discussed.

Caiman crocodylus hemoglobin. Complete primary structures of alpha and beta chains; phylogenic and regulatory aspects.

In some reptiles, the hemoglobin oxygen affinity is lowered by CO2 and not by the usual phosphate cofactors. To understand the molecular mechanism of this regulation, Caiman crocodylus hemoglobin's primary structure has been determined. The alignment of the 141 residues of the alpha chain as well as the 146 of the beta chain were obtained by classical method of sequence analysis and are compared with some mammalian, avian, amphibian, and fish hemoglobin chains. Furthermore, from these sequences, it is possible to explain the non-interaction of phosphorylated cofactors with the caiman deoxyhemoglobin on the basis of mutations of the amino acids responsible for their fixation on the beta chain. Among these residues only lysine beta 82 is unchanged. In addition a site has been proposed for the fixation of HCO3- which involves ionic bonds with serine beta 1 and glutamic acid beta 144 (Perutz et al. (1981) Nature 291, 682-684) (1).

Reoxidation of reduced hen egg white lysozyme fragment 1-123.

The reactivation of reduced lysozyme, whose 6 COOH-terminal amino acid including cysteine 127 were cut off, was studied. The results show that the disulfide bridge I-VIII as well as the COOH-terminal hexapeptide do not play a decisive role in the acquisition of the native 3-dimensional structure of the enzyme.