SUBCELLULAR LOCALIZATION, PURIFICATION AND PROPERTIES OF A CDP-DIACYLGLYCEROL - DEPENDENT PHOSPHATIDYLSERINE SYNTHASE IN BACILLUS LICHENIFORMIS

A CDP-diacylglycerol dependent phosphatidylserine synthase was detected in three species of gram-positive bacilli, viz. Bacillus licheniformis, Bacillus subtilis and Bacillus megaterium; the enzyme in B. licheniformis was studied in detail. The subcellular distribution experiments in cell-free extracts of B. licheniformis using differential centrifugation, sucrose gradient centrifugation and detergent solubilization showed the phosphatidylserine synthase to be tightly associated with the membrane. The enzyme was shown to have an absolute requirement for divalent metal ion for activity with a strong preference for manganese. The enzyme activity was completely dependent upon the addition of CDP-diacylglycerol to the assay system; the role of the liponucleotide was rigorously shown to be that of phosphatidyl donor and not just a detergent-like stimulator. This enzyme was then solubilized from B. licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue-dextran Sepharose. The purified preparation showed a single band with an apparent minimum molecular weight of 53,000 when subjected to SDS polyacrylamide gel electrophoresis. The preparation was free of any phosphatidylglycerophosphate synthase, CDP-diacylglycerol hydrolase and phosphatidylserine hydrolase activities. The utilization of substrates and formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential BiBi reaction as opposed to the ping-pong mechanism exhibited by the well studied phosphatidylserine synthase of Escherichia coli. Proteolytic digestion of the enzyme yielded a smaller active form of the enzyme (41,000 daltons) which appears to be less prone to aggregation.^ This has been the first detailed study in a well-defined bacillus species of the enzyme catalyzing the CDP-diacylglycerol-dependent formation of phosphatidylserine; this reaction is the first committed step in the biosynthetic pathway to the major membrane component, phosphatidylethanolamine. Further study of this enzyme may lead to understanding of new mechanisms of phosphatidyl transfer and novel modes of control of phospholipid biosynthetic enzymes. ^

INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

SYNTHETIC PROBES FOR STUDYING TUFTSIN-RECEPTOR INTERACTIONS ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Tuftsin is an immunopotentiating tetrapeptide of the sequence L-Thr-L-Lys-L-Pro-L-Arg with anti-microbial and anti-tumor enhancing capabilities. These enhancing functions are manifested through the host's granulocytes and monocytes. In delineating tuftsin's mechanism of action, both radiolabeled and fluorescent probes were synthesized. The radiolabeled probe of tuftsin, L-proly-3,4-('3)H(N) -tuftsin, was obtained through the synthesis and subsequent catalytic hydrogenation of L-3,4-dehydroprolyl ('3)-tuftsin using tritium gas. This procedure yielded a probe with a specific activity of 44.9 Ci/mmole. This radiolabeled probe of tuftsin was used in competitive inhibition studies with tuftsin, the tuftsin analogues Lys-Pro-Arg, Thr-Lys-Pro-Arg(NO(,2)) and (DELTA)('3)-pro('3) -tuftsin as well as with the chemotactic peptide f-Met-Leu-Phe. From the competitive binding curves, the K(,D) for tuftsin was estimated to be 80 nM, a value that approaches the concentration of tuftsin that evokes a half maximal biological response. The approximate Ki's for the tuftsin analogues (33 nM) approached that of tuftsin itself (40 nM). On the other hand, approximately a two log difference in the Ki was seen with the chemotactic tripeptide, indicating that tuftsin may indeed be acting through the chemotactic peptide receptor. This conclusion is further strengthened by studies using an N-terminal derivitized mono-fluoresceinated tuftsin probe and image intensification microscopy. These studies showed that like the chemotactic peptide, tuftsin initially binds to diffusely distributed receptors on the surface of human granulocytes. The tuftsin-receptor complexes then rapidly redistribute to form patches (5 min @ 37(DEGREES)C) which are then internalized. Whether redistribution and internalization of tuftsin-receptor complexes is crucial in effecting a biological response, or simply an intermediary point leading ultimately to degradation, is still not clear. This process, however, may provide the target cell with an early time point in modulating the biological effects of tuftsin through down-regulation of cell surface receptor sites. ^

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^