Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.     

Water and solute transport by the Malpighian tubules of the stick insect,Carausius morosus

Summary Structural features of the principal, urine-secreting cells (type 1 cells) of the Malpighian tubules of Carausius are de scribedquantitatively and discussed in relation to possible mechanisms of water and solute transport. Mitochondria are arranged in two bands of about equal volume near to the basal and apical surfaces, suggesting active processes occur at both surfaces. Basal infoldings and apical microvilli which greatly amplify the cell surface are probably primarily devices to increase the passive permeability of the tissue to solutes. They do not provide functionally significant standing-osmotic-gradients. The extensive endoplasmic reticulum is locally differentiated into several components and ramifies between the infoldings and along microvilli but probably is not an intracellular conduit for the majority of urinary constituents. Vesicles and stages in their formation or liberation are observed both basally and apically although they probably do not contribute significantly to transcellular transport. At present it remains a problem to satisfactorily account for observations that the urine of Carausius can be hypotonic.This investigation formed part of a dissertation for the degree of Ph. D. in the University of Newcastle upon Tyne. It is a pleasure to thank Prof. J. Shaw for his advice and encouragement and the Science Research Council for financial support.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: evidence for mutant forms of folylpolyglutamate synthetase

A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine (CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 (one spontaneous and one Pt(S0₄)₂ induced) have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation (37 °C, pH 7.4 or 9.0) than the parent CHO enzyme. Increased sensitivity of revertant FPGS is observed irrespective of whether one assays the specific catalysis of radioactive tetrahydropteroyldi- or tetraglutamate synthesis. ATP and MgCl₂ protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. AUXB3 FPGS shows a normal sensitivity to 37 °C heat inactivation, but it has an altered substrate saturation and specificity pattern when assayed for tetrahydropteroyldi[U-¹⁴C]glutamate synthesis. Also, unlike the FPGS from parent CHO and a genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra[U-¹⁴C]glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions.     

Aerobic photolysis of alkylcobalamins: quantum yields and light-action spectra

Quantum yields (φ) for the aerobic photolysis of 5′-deoxyadenosylcobalamin (dAB₁₂), methylcobalamin (MeB₁₂), propylcobalamin (PrB₁₂), and ethylcobalamin (EtB₁₂) were determined as a function of the irradiation wavelength. φ Determinations were made for both the base-on and base-off forms of each compound (except base-off dAB₁₂) at incident wavelengths from 250 nm to 570 nm. As a rule, the φs were high (0.1–0.5) and they varied significantly with respect to the irradiation wavelength. In general, each alkylcobalamin at pH 7.0 displayed a quantum yield spectrum distinct from its base-off form at pH 1.0. Across most of the spectrum, the φs of the base-off form were appreciably smaller than the base-on φs of the same compound. An exception to this generality was MeB₁₂ for which the φs at pH 1.0 were about the same as, or slightly greater above 450 nm than those at pH 7.0. At pH 7.0 and in the visible region the trend of the φs was dAB₁₂ < MeB₁₂ < PrB₁₂ < EtB₁₂. Under neutral conditions each compound showed a broad quantum yield peak in the 450–470 nm region.From the quantum yield and absorption spectra, photolysis spectra were calculated for 5.0 × 10^?5m solutions of each compound. The light-action spectra accurately give the relative rates/μ Einstein that these solutions photolyze at each wavelength. Thus, for example, MeB₁₂ photolyzed faster at pH 7.0 versus pH 1.0 in 510 nm light, but it photolyzed slower at pH 7.0 versus pH 1.0 in 450 nm light. Solutions of each compound photolyzed faster in the ultraviolet region as opposed to the visible (e.g., 310 nm versus 510 nm).Our findings show that the previously reported photolysis rates estimated by others with tungsten lamps provide no valid information about the intrinsic photolability of various alkyl-cobalt bonds. This also applies to the relative white-light photolysis rates reported for the base-on versus the base-off form of MeB₁₂. All such relative rates are artifacts which represent only the extent of overlap between the true action spectrum and the light emission spectrum of an incandescent lamp.     

The EPR of low spin heme complexes. Relation of the t2g hole model to the directional properties of the g tensor, and a new method for calculating the ligand field parameters.

A simple method is presented for calculating the parameters of the hole model for distorted octahedral, low spin (t2g)5 complexes. In the case of negligible covalent bonding explicit formulas for the coefficients of the Kramer's doublet, (formula: see text). The two numerically largest g values define the plane and orientation of the orbital with the largest coefficient, which in turn indicates the directions of maximal unpaired spin density. The energy of eta with respect to xi (in units of lambda, the spin-orbit coupling constant) is (formula: see text). The product gzgygx, independent of axes, and positive for free electrons, is shown to be positive for tetragonal and negative for nearly octahedral complexes. It is considered positive for hemes. In this method coefficients will only be normalized when there is no covalency. For the majority of published cases they are, to about 1%. Since this discrepancy is larger than can be caused by propagated errors, covalency must be the rule. For comparative purposes A and B, uncorrected for covalency, should still be useful. Examination of published complete g tensors for five hemes shows that the largest g value is nearly normal to the heme plane. If the g values are taken positive and labelled so that gz greater than gy greater than gx, then the proper tetragonal axis is roughly normal to plane of the ring in hemes, but not in chlorins.     

Genes for serum amyloid A proteins map to Chromosome 7 in the mouse

Summary Several restriction fragment length variants have been detected among inbred strains using a mouse serum amyloid A cDNA clone. Five variants were shown to segregate as a single genetic unit and were mapped to Chromosome 7 between the glucose phosphate isomerase locus (Gpi-1) and the pink eye dilution locus (p) using recombinant inbred and congenic strains. The finding that no major MspI or BclI restriction fragments were shared between digests of DNAs from a Chromosome 7 congenic strain and its inbred partner, indicate that most, and probably all, sequences detected with the probe are clustered on Chromosome 7. Aneuploid mapping was used to show that the serum amyloid A gene complex (Saa) is proximal to the Chromosome 7 breakpoint in T(7;X)1Ct, a translocation in which the middle third of Chromosome 7 is inserted into the X-chromosome. A survey of inbred strains revealed a single common Saa haplotype and eight rare haplotypes. The complex distribution of 14 different variants suggests that recombination may have played a role in haplotype evolution.This work was supported by grants GM18684 and CA33093 from the National Institute of General Medical Sciences and the National Cancer Institute, respectively.     

Antagonistic and synergistic interactions between aluminum and manganese on growth of Vigna unguiculata at low ionic strength

Concentrations of soluble aluminum (Al) and manganese (Mn) frequently reach phytotoxic levels in acid soils. While dose response relationships for these metals are well documented, the effects of combined exposure have received less attention. We have examined the effect of combinations of Al and Mn on growth and metal accumulation in Vigna unguiculata (L.) Walp. grown in solution culture under conditions of low ionic strength (conductivities typically < 100 µS cm⁻¹). The nature of interaction between these metals varied with the specific physiological response, the part of the plant investigated, and the relative amount of stress imposed. Analysis of growth data provided evidence for amelioration of metal toxicity (antagonistic effects), although this effect was dose dependent. Analysis of metal content data provided evidence for antagonistic and synergistic (exacerbation of toxicity) effects, again depending on dose. Analysis of foliar symptoms also provided evidence for antagonisms and synergisms, with the nature of the response dependent on the specific physiological response and specific plant part investigated. In contrast with previous reports, evidence for antagonistic, synergistic, and multiplicative effects on growth, metal uptake, and expression of foliar symptoms have been obtained under physiologically and environmentally relevant conditions. These results suggest a more detailed analysis of the potential for interactions between metals in the environment is required.