Expression of cloned calf prochymosin gene sequence in Escherichia coli

An expression plasmid for calf prochymosin (prorennin) cDNA was constructed. The plasmid (pCR301) contains the lacUV5 promoter in front of the fused gene in which the codons for the N-terminal four amino acids of prochymosin cDNA were replaced with those for the N-terminal ten amino acids of beta-galactosidase. Synthesis of the fused protein with the expected Mr was detected immunologically in Escherichia coli harboring pCR301. The product seemed to be localized in the cell membrane of the bacterial host.     

Expression of recombinant calf prochymosin in mammalian cell culture.

The calf preprochymosin cDNA was cloned into an extrachromosomal mammalian cell expression vector containing Epstein-Barr virus sequences using polymerase chain reaction. Transfection of HeLa cells yielded Hygromycin B resistant cell clones, expressing immunoreactive prochymosin, which was quantitatively secreted into the culture medium. Based on Western blotting we estimated that selected cell clones produced about 10-20 mg prochymosin per liter in 20 h. The biological activity of the secreted chymosin was confirmed by milk clotting assay.     

Unusual zymogen-processing properties of a mutated form of prochymosin

Site-specific mutagenesis of the gene encoding bovine prochymosin was used to produce a mutated zymogen in which seven contiguous amino acids of the N-terminal propeptide had been deleted and an eighth residue had been substituted. This altered region spans the normal site of autocatalytic proteolysis that occurs at the same time as (enzymatic) activation of prochymosin at acidic pH. Activation of the mutated zymogen at pH 4.5 was extremely slow, and cleavage occurred at an unusual Ser-Lys bond in the propeptide of the zymogen. The mutated prochymosin incubated at pH 2 generated the usual pseudochymosin by cleavage of the normal Phe-Leu bond, but at a rate severalfold slower than the authentic zymogen. These results indicate that even after deletion of seven of 42 amino acids of the propeptide the mutant protein could still assume a prochymosin (zymogen) structure, although these changes did result in striking differences in acid-catalyzed activation and processing reactions at one but not the other of the two processing sites of prochymosin.     

Synthesis and properties of PNA oligomers containing orotic acid derivatives

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.     

Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter

To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300 000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lacUV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.     

Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives

alpha-Tocopherol and alpha-tocopheryl succinate are biologically active lipids. The activity of these lipids may be related to how they affect membrane physical-chemical properties. Utilizing fluorescence methods, we have investigated the effect of alpha-tocopherol, alpha-tocopheryl succinate, and alpha-tocopheryl acetate on the properties of model membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In liquid-crystalline phase phospholipid bilayers, alpha-tocopherol decreased acyl chain mobility and decreased the interfacial polarity, but had no effect on the interfacial surface charge. In contrast, alpha-tocopheryl succinate had little effect on acyl chain motion or interfacial hydration, but increased the interfacial surface charge. alpha-Tocopheryl acetate had very little effect on any of the measurements of these bilayer properties. In a gel phase bilayer, alpha-tocopherol decreased acyl chain order, whereas alpha-tocopheryl succinate and alpha-tocopheryl acetate did not. Each alpha-tocopheryl derivative had a different effect on interfacial polarity, however, only alpha-tocopheryl succinate increased the interfacial surface charge. The acylation of alpha-tocopherol abolishes its antioxidant activity and generates molecules with different membrane physical properties. The non-polar acetate group of alpha-tocopheryl acetate locates this compound in a region of the bilayer where it has little effect on bilayer interfacial properties. The free carboxyl group of alpha-tocopheryl succinate is located in the interfacial region of the bilayer where it increases the membrane surface charge.     

The anion recognition properties of hydrazone derivatives containing anthracene

A series of artificial receptors, hydrazone derivatives containing anthracene, have been designed and synthesized. The interaction of these receptors with biologically important anions was determined by UV–vis, fluorescence and ¹H NMR titration experiments and theoretical investigation. Results indicate that the receptor (1) without NO₂ shows no binding ability for various anions. The other receptors (2 and 3) show the highest binding ability for acetate (AcO⁻) among studied anions (fluoride (F⁻), dihydrogen phosphate (H₂PO₄⁻), chloride (Cl⁻), bromide (Br⁻), iodide (I⁻)); and the binding ability for AcO⁻ is not interfered by the existence of other anions. The additions of AcO⁻, F⁻ and H₂PO₄⁻ can arouse different degrees of fluorescence quenching. ¹H NMR titration shows that the interaction between the receptor 2 and F⁻ firstly depends on the hydrogen-bond formation; later the interacted site NH is deprotonated and the added F⁻ forms hydrogen bond with the near CH in Schiff base. Moreover, visual color changes accompany guest binding, enabling this system to act as colorimetric anion sensors.     

Molecular cloning and expression in yeast of caprine prochymosin

We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.     

Solvent effects on fluorescence properties of protochlorophyll and its derivatives with various porphyrin side chains

Fluorescence spectra and fluorescence lifetimes of protochlorophyll (Pchl) were measured in organic solvents having different physical and chemical properties and were analyzed taking into account the nonspecific (dependent on bulk solvent parameters), and specific (e.g. H bonds, Mg coordination) solvent–solute interactions. The energy of the fluorescence emission band decreased, while the Stokes shift increased for increasing solvent orientation polarizability, which is a function of both the dielectric constant (ε) and the refractive index (n). The extent of the dependence of the Stokes shift on solvent orientation polarizability was higher in protic (i.e. those able to form hydrogen-binding) than in aprotic solvents. High value of the Stokes shift was also observed in pyridine and methanol, i.e. in solvents hexacoordinating the central Mg atom. The fluorescence decay of Pchl was monoexponential in all of the investigated solvents. The fluorescence lifetime decreased for increasing solvent orientation polarizability from 5.5 ± 0.1 ns in 1,4-dioxane to 3.3 ± 0.1 ns in methanol. Longer lifetime values were observed in the case of aprotic solvents than in protic solvents. The hexacoordination of Mg had no effect on the fluorescence lifetime. The present data are discussed with respect to results found for protochlorophyllide (Pchlide) (My?liwa-Kurdziel et al. in Photochem Photobiol 79:62–67, 2004), and they indicate that the presence of phytol chain in the porphyrin ring influences the spectral properties of the whole chromophore. This is the first complex analysis comparing the fluorescence emission and fluorescence lifetimes of purified Pchl and Pchlide.     

Expression and binding properties of a soluble chimeric protein containing the N-terminal domain of the Duffy antigen

The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fy(a) and Fy(b), is a promiscuous chemokine receptor. In this report we describe the expression and purification of a chimeric protein composed of the amino-terminal extracellular domain of the Duffy antigen (aa 3-60), C-terminal intracellular fragment of glycophorin A (GPA, aa 104-131), and the hexahistydyl tag. We obtained two forms of the recombinant protein containing the Fy(a) or Fy(b) epitope, denoted Fy(a)/GPA and Fy(b)/GPA, respectively. These constructs were expressed in Escherichia coli as periplasmic proteins and were purified by affinity chromatography on the Ni-NTA-agarose. Both proteins bound the monoclonal antibodies recognizing the common Fy6 epitope of the Duffy antigen and an epitope of the C-terminal fragment of GPA, and only the Fy(a)/GPA bound anti-Fy(a) antibody. However, binding of IL-8 to the recombinant proteins was not detected, which indicated that an N-terminal domain of the Duffy antigen is not sufficient for an effective chemokine binding. The lack of the chemokine binding was not likely to be due to the lack of glycosylation of the Fy/GPA, since IL-8 was effectively bound to de-N-glycosylated erythrocytes.     

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the GC sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the AT and AU sequences. GC base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to AT base pairs. On the contrary, the continuous ribonucleotide GC sequence binds the phenazines more weakly than does the AU sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.     

Dyeing and diffusion properties of modified novel cellulose with triazine derivatives containing cationic and anionic groups

Cellulose is chemically modified with the compounds containing cationic and anionic groups. Dyeing and diffusion properties of modified cellulose are discussed. The exhaustion and fixation of reactive dyes on modified cellulose are higher than those on unmodified cellulose. Compared with unmodified cellulose, the dyed modified cellulose also gets good washing fastness. The diffusion coefficients of dyes at different temperature are calculated. Compared with unmodified cellulose, the diffusion of dyes in the modified cellulose shows significant change.     

Cyclic enkephalin analogs containing various para-substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists.

The cyclic enkephalin analog H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) is a highly potent opioid agonist with IC(50)s of 35 pm and 19 pm in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe(1)-analog of this peptide showed 370-fold and 6790-fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr(1) hydroxyl-group in the interaction with mu and delta opioid receptors. In the present study, the effect of various substituents (-NH(2), -NO(2), -CN, -CH(3), -COOH, -COCH(3), -CONH(2)) introduced in the para-position of the Phe(1)-residue of H-Phe-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) on the in vitro opioid activity profile was examined. Most analogs showed enhanced mu and delta agonist potencies in the two bioassays, except for the Phe(pCOOH)(1)-analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH(3))(1)- and the Phe(pCONH(2))(1)-analogs. The latter compound showed subnanomolar mu and delta agonist potencies and represents the most potent enkephalin analog lacking the Tyr(1) hydroxyl-group reported to date. Taken together, these results indicate that various substituents introduced in the para-position of Phe(1) enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure-activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Synthesis of N-hydroxysuccinimide esters of phosphatidylethanolamine and some properties of liposomes containing these derivatives

The N-hydroxysuccinimide (NHS) ester of N-suberyl-dimyristoylphosphatidylethanolamine (sub-DMPE) was synthesized by reaction of DMPE with disuccinimidyl suberate, and isolated by preparative plate chromatography. Liposomes, which contain NHS-sub-DMPE, can covalently bind compounds that possess a free amino group such as ε-dinitrophenyl-lysine. The extent of DNP-lysine binding is influenced by the time and temperature of incubation, the amount of NHS-sub-DMPE incorporated into the liposomes, and the initial concentration of DNP-lysine. Binding occurs as a consequence of the formation of a new dinitrophenylated compound which has been characterized. Although NHS-sub-DMPE is stable to storage in organic solvents, preformed liposomes rapidly lose their ability to bind DNP-lysine due to hydrolysis of the N-hydroxysuccinimide ester bond. These findings bear on the future applicability of liposomes, containing N-hydroxysuccinimide esters of PE, as illustrated by the preparation of immunogenic liposomes.     

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.     

Study of effect of water on the interaction of DNA and actinocin derivatives with various aminoalkyl chain length by infrared spectrophotometry and computer modeling

A comparative study of the effect of water on the interaction of DNA with actinocin derivatives having different numbers of methylene groups in side chains was performed by IR spectroscopy. It was found that, as relative humidity increases, water molecules simultaneously bind to hydrate-active sites of DNA and ligands. The absorption band at v = 1137 cm-1, caused by oscillations of the C-O and P-O groups of atoms in the DNA-ligand complex having two methylene groups, is due to the interactions between the cationic groups of the ligand and the sugar-phosphate backbone of DNA, which may be one of the reasons for the high stability of this complex. Using computer simulation of interaction of DNA fragments and actinocin derivatives in water environment, molecular models of the formation of their complexes for two ways of binding were constructed.     

Investigations on the activation of bovine prochymosin.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.