Chemical transformation of eremofortin C into PR toxin.

The natural products of both eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Because the chemical structures of EC and PR toxin are closely related to each other and differ only by a hydroxyl functional group in EC and an aldehyde functional group in PR toxin at the C-12 position, the chemical transformation of EC into PR toxin was investigated. Oxidation with a chromic anhydride-pyridine complex was found to be the most satisfactory method.     

Control of sugar transport in human fibroblasts independent of glucose metabolism or carrier-substrate interaction

Transport regulation by different metabolizable and nonmetabolizable sugars was studied in human fibroblasts. Sugars were classed as glucose-like (D-mannose, 3-0-methyl-D-glucose, thio-D-glucose, and D-allose) and starvation-like (D-galactose, D-fructose, L-glucose, D-xylose, 6-deoxy-D-glucose and 2-deoxy-D-glucose) based on their competence in curbing glucose starvation enhanced transport. No significant correlation existed between the ability of a sugar to curb hexose transport and the KI of that sugar in inhibiting hexose transport. Independence of the transport curb from glucose metabolism was observed since nonmetabolizable analogs of D-glucose when substituted for D-glucose in the culture medium effected glucose [i.e. 3-0-methyl-D-glucose (3-OMG)] and starvation-like (i.e. 6- and 2-deoxy-D-glucose) effects. The KI of inhibition pf 2-deoxy-D-glucose transport for 3-OMG was 8.5 mM, similar to those obtained for 6-deoxyglucose and 2-deoxyglucose on 2-deoxyglycose transport (7.5 and 3.5 mM, respectively) and on 3-0-methylglucose transport (3.5 and 2.5 mM, respectively). An equimolar mixture of D-glucose and 3-OMG (5.55 mM each) was more effective than 11.1 mM D-glucose or 3-OMG alone in curbing hexose transport or reversing hexose starvation induced increases in transport. The effect of 3-OMG may be independent of glucose metabolism but it is possible that 3-OMG structurally mimics a metabolite of glucose that may interact with intracellular regulators of carrier degradation and or expression.     

On the structure of heme d1. An isobacteriochlorin derivative as the prosthetic group of dissimilatory nitrite reductase?

Timkovich and co-workers have recently proposed a chlorin macrocycle structure for the heme d1 prosthetic group isolated from cytochrome cd1 of Pseudomonas aeruginosa and Paracoccus denitrificans (Timkovich, R., Cork, M. S., and Taylor, P. V. (1984) J. Biol. Chem. 259, 1577-1585; 15089-15093). However, this chlorin structure deduced by them is not entirely consistent with the spectral data. An alternative structure is proposed here based on the available spectral evidence. It is suggested that heme d1 in vivo is not a chlorin, but a dioxo-isobacteriochlorin having two adjacent pyrrole rings saturated.     

Formation of an infinite beta-sheet arrangement dominates the crystallization behavior of lambda-type antibody light chains

The packing interactions in crystals of human lambda-type antibody light chain dimers have been reviewed. These homologous proteins are composed of individually specific variable domains, but all have very similar constant domain sequences. The proteins do not emulate each other in their overall crystallization behavior: each attains an individually characteristic space group or unit cell dimensions. However, each of these protein crystals has one unit cell dimension in common, 72.4(+/- 0.2) A. Examination of the protein packing in these crystals reveals that the common cell dimension is a consequence of a packing arrangement of their constant domains, which is conserved in all three crystals. In this striking arrangement, beta-sheets of adjacent constant domains are placed in juxta-position to form an "infinite chain". Although this constant domain packing pattern is rigorously conserved, the variable domain packing arrangements in each of these crystals are different. The conservation of the "infinite" beta-sheet pattern suggests that the constant domain interactions dominate the thermodynamic energy of lattice formation, probably through a combination of specific hydrogen bond formations and by a decrease in the solvent-accessible surface. A single amino acid substitution prohibits this characteristic interneighbor hydrogen bond pattern in the homologous kappa-type light chains. This may explain the observation that very few kappa-type light chains have been crystallized.     

Cholecystokinin receptor mediated hydrolysis of inositol phospholipids in guinea pig gastric glands

CCK-octapeptide (CCK-8) (EC50 = 0.5 nM), in the presence of Li+, increased 3H-inositol phosphate (IP) accumulation in guinea pig gastric glands prelabeled with 3H-inositol. CCK-8 desulfate, human gastrin I and pentagastrin were much less potent than CCK-8. Antagonists of CCK receptors such as proglumide, dibutyryl-c-GMP and CBZ-Tyr (SO3H)-Met-Gly-Trp-Met-AspNH2 shifted the CCK dose response curve to the right. However, histamine (H1 and H2), cholinergic, substance P and alpha- and beta-adrenergic receptor antagonists had no effect on 3H-IP accumulation induced by CCK. The results suggest that CCK receptor activation in gastric glands leads to an enhanced breakdown of inositol phospholipids which may relate to calcium mobilization and pepsinogen secretion.     

Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens

The binding of biologically active 125I-Bolton-Hunter (BH)-NPY to rat brain membranes was saturable and reversible and regulated by inorganic cations and guanyl nucleotides consistent with other neurotransmitter receptor systems. The concentration of specific 125I-NPY binding differed in various brain regions, being highest in the hippocampus and lowest in the cerebellum. Scatchard analysis of 125I-NPY binding showed a single class of receptor sites with a Kd = 0.1 nM and Bmax of 3 pmole/g tissue in hippocampus. Peptide YY, porcine and human NPY inhibited the specific 125I-BH-NPY binding with IC50 values of 50-120 pM. In contrast, human NPY free acid and pancreatic polypeptides from human (HPP), rat (RPP) and avian (APP) sources were much weaker (IC50 greater than or equal to 300 nM). The rank order of potencies for NPY analogs and the inactivity of APP and HPP fragment (31-36) on brain binding appeared to correlate with their relative activities in inhibiting contractions of the field-stimulated rat vas deferens. However, PYY, HPP and RPP exhibited activity in the field-stimulated rat vas deferens indicative of a possible action upon sites distinct from the brain NPY binding site.     

Nuclear, Virescent Mutants of Zea mays L. with High Levels of Chlorophyll (a/b) Light-Harvesting Complex during Thylakoid Assembly

We have found nuclear, recessive mutants in Zea mays L. where assembly of the major chlorophyll (a/b) light-harvesting complex (LHC) was not delayed relative to most other thylakoid protein complexes during thylakoid biogenesis. This contrasts with the normal development of maize chloroplasts (NR Baker, R Leech 1977 Plant Physiol 60: 640-644). All four mutants examined were allelic and virescent, and displayed visibly higher yields of leaf Chl fluorescence during greening. Fully greened mutants had normal leaf Chl fluorescence yield and normal levels of LHC, and grew to maturity under field conditions. Therefore, delayed LHC assembly is not an obligate feature of thylakoid differentiation.

Assigning the molecular basis for the mutation should provide information concerning reguation of LHC assembly. Several possibilities are discussed. The pleiotropic mutant phenotype is not attributable to defects in thylakoid glycerolipid synthesis. Thylakoids isolated from greening mutant leaf sections had elevated glycerolipid/Chl ratios. In addition, both the molar distribution and acyl composition of four major glycerolipids were normal for developing mutant thylakoids.

     

Evidence that the outer membrane protein gene nmpC of Escherichia coli K-12 lies within the defective qsr'' prophage.

Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent. The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2. Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein. When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene. Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene. These results were supported by blot hybridization analysis of the E. coli chromosome with probes derived from the lc gene of phage PA-2. A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence. We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element.     

Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase

6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have characteristics similar to those of native and pyridoxal enzymes, respectively, except that the former two enzymes have lower Vmax values. 19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme forms a neutral enolimine Schiff base. Because the UV and fluorescence spectra of 6-FPLP phosphorylase are comparable to those obtained with native phosphorylase, it further confirms the postulate that pyridoxal phosphate forms a neutral enolimine Schiff base in phosphorylase. The results suggest that the 3-OH group is protonated and the pyridine nitrogen unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study of 6-FPLP- and 6-FPAL-reconstituted phosphorylases in the inactive and active states indicates that the protein structure near the coenzyme binding site undergoes certain changes when these enzymes are activated by the substrates and AMP. The comparison of the properties of 6-FPLP-reconstituted and native phosphorylases implies that the ring nitrogen of the coenzyme PLP in phosphorylase may interact with the protein during catalysis, and this interaction is important for efficient catalysis by phosphorylase.     

Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.