Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis.

During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermatozoa. During this process there occurs remodeling of cell structure and loss of most of the cytoplasm and a large fraction of cellular proteins. To evaluate the role of the ubiquitin proteolytic system in this protein loss, we measured levels of ubiquitinated proteins and rates of ubiquitin conjugation in extracts of testes from rats of different ages. Endogenous ubiquitin-protein conjugates increased till day 30 and then reached a plateau. In parallel, there was a progressive increase in the rate of conjugation of ubiquitin to proteins in testis extracts from these animals. To test the importance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extracts was carried out and the remaining conjugation activity in supernatants was assayed. Depletion of either enzyme family resulted in decreased conjugation. However, most of the conjugation activity and, more importantly, the increased conjugation during development were UBC4-dependent. Immunocytochemistry demonstrated a marked increase in expression of UBC4 in spermatids, consistent with the UBC4-dependent activation of conjugation seen in vitro. In situ hybridization studies evaluated the contribution of various UBC4 isoforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 mRNA was restricted to germ cells with high levels of expression in round and elongated spermatids. UBC4-testis had previously been shown to be expressed only in spermatids. Our data suggest that induction of various UBC4 isoforms activates overall conjugation and plays an important role in the cellular remodeling and protein loss occurring during spermatogenesis.     

Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.     

Prostaglandin-sepharose: Application to the purification of bovine lung 15 (S) -hydroxyprostaglandin dehydrogenase

Prostaglandins E₁, F_1α and A₂ were covalently joined to the surface of Sepharose as carboxamide linkages. The insolubilized prostaglandins were shown to function effectively in the purification of 15(S) -hydroxyprostaglandin dehydrogenase.     

Block of axoplasmic transport in vitro by vinca alkaloids

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1–100μM was examined. The relative order of potency in blocking axoplasmic transport was VCR > VLB > VDS at a concentration of 25μM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block ot the transport mechanism is discussed.     

Initial synthesis of globin peptide chains in differentiating embryonic red blood cells

The initial time of synthesis of globin polypeptide chains in differentiating red cells has been delineated. Three analytical techniques were used, namely, SDS-polyacrylamide gel electrophoresis, acid urea polyacrylamide gel electrophoresis, and two-dimensional gel electrophoresis. The results indicate that globin peptide chains are synthesized in proerythroblasts at the initial time when globin mRNA first enters into the cytoplasm, and there is no apparent time gap between these two events.     

Toxic shock syndrome in a patient with systemic lupus erythematosus.

A case is presented of toxic shock syndrome in a patient with systemic lupus erythematosus. Toxic shock syndrome is rarely reported in patients who are immunosuppressed, perhaps because such patients are often treated vigorously with antibiotics at the earliest sign of infection. The association in this case may have been coincidental.     

Sequential passage of alpha2mu-globulin through the hepatic endoplasmic reticulum and Golgi apparatus during secretion.

Studies on the synthesis and secretion of the sex-dependent urinary protein, alpha2mu-globulin, have been extended by establishing its sequential passage from the rough to the smooth endoplasmic reticulum and Golgi-rich fractions of the liver of adult male rats. After injection of 14C-labeled amino acids, the maximum radioactivity of alpha2mu occurred at 20 min in the rough, 25 min in the smooth microsomes and 30 or 35 min in the Golgi-rich fractions. Radioactive alpha2mu-globulin appeared in the bloodstream and kidneys after a lag of 20--25 min. Results indicate that alpha2mu-globulin follows a secretory pathway similar to that of serum albumin.