Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.     

Streptomycin- and rifampin-resistant mutants of Escherichia coli perturb F exclusion of bacteriophage T7 by affecting synthesis of the F plasmid protein PifA.

Certain alleles of rpsL that confer resistance to the antibiotic streptomycin almost completely relieve F exclusion of bacteriophage T7. Introduction of a specific rpoB allele conferring resistance to rifampin into the rpsL strain restores the ability of the F-containing strain to exclude T7. This variation in the severity of F exclusion is reflected in the levels of the F-encoded inhibitor protein PifA: F'-containing cells that harbor specific rpsL alleles are phenotypically Pif-, but become Pif+ by the further acquisition of a specific rpoB allele. F-containing cells harboring the gyrA43(Ts) mutation also appear phenotypically Pif-, possibly because repression of the pif operon is enhanced by an altered DNA conformation in the gyrase mutant strain.     

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

Phosphorylation of hydroxyproline in a synthetic peptide catalyzed by cyclic AMP-dependent protein kinase.

The cyclic AMP-dependent protein kinase catalyzes the phosphorylation of hydroxyproline present in the heptapeptide, Leu-Arg-Arg-Ala-Hyp-Leu-Gly. The Km value for the reaction with this substrate was high (approximately 18 mM) compared to the Km values reported for the analogous threonine and serine-containing peptides, which were 0.59 mM and 0.016 mM, respectively (Kemp, B.E., Graves, D.J., Benjamini, E., and Krebs, E.G. (1977) J. Biol. Chem. 252, 4888-4894). The Vmax value with the hydroxyproline-containing peptide was 1 mumol . min-1 mg-1 in contrast to Vmax values of 6 mumol . min-1 mg-1 and 20 mumol . min-1 mg-1 for the threonine- and serine-containing peptides, respectively. Phosphate esterified to hydroxyproline present in the peptide was relatively stable in hot alkali, only 10% being released as Pi within 30 min in 0.1 N NaOH at 100 degrees C, whereas all of the phosphate was released from the phosphoserine peptide analogue under these conditions. Phosphohydroxyproline in the peptide was also more stable to acid (5.7 N HCl, 110 degrees C) than phosphoserine, the time for 50% release as Pi being 15 h in contrast to 6 h for the latter.     

A comparison of viable counts and adenine nucleotide analysis to determine the effect of antimicrobial agents on dental plaque

The effects of three antimicrobial agents on smooth surface dental plaque in monkeys were assessed using a plaque index, bacterial viable count, and adenine nucleotide analysis. The effects of the agents were revealed equally well when, viability was assayed by extractable adenosine triphosphate (ATP) or conventional viable cell counts. A persistent effect of one of the agents was revealed by both viable count and extractable ATP. These interpretations were supported by calculations of adenylate energy charge from the adenine nucleotide content of the smooth surface dental plaque samples.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the brain and infundibulum of the sheep

Summary To determine how neural influences control the function of the pineal gland, morphological and biochemical relationships after pharmacological treatment have been studied in rat pineal cells in monolayer cultures. Norepinephrine (NE) and dibutyryl cyclic 3,5-adenosine monophosphate (dBcAMP) treatment of cells that had been in culture for 5 and 21 days produced a stimulation in the enzyme activity of serotonin N-acetyl transferase, an enzyme important in indole synthesis. NE and dBcAMP also produced morphological changes which were dependent on the time of cells in culture. When 5 day-cultures were treated with NE and dBcAMP, light and dark cells were noted and endoplasmic reticulum increased and became more organized. Only dBcAMP treatment at 5 days produced an increase in dense granules and an elongation of cytoplasmic processes. Treatment of 21 day-cultures with dBcAMP also produced an increase in cytoplasmic processes while treatment with NE produced an increase in the synaptic ribbons and clear vesicles within the processes.     

Phosphorylation of a synthetic gastrin peptide by the tyrosine kinase of A431 cell membranes

The peptide Arg.Arg.Leu.Glu.Glu.Glu.Glu.Glu.Ala.Tyr.Gly was synthesized as an analogue of residues 20–30 of human gastrin 34. The epidermal growth factor-stimulated tyrosine kinase of A431 cell membranes phosphorylated the peptide's single tyrosine residue. K_m values of 0.11 and 0.61mM and V_max values of 1.71 and 0.68nmol/min/mg were obtained in the presence and absence of epidermal growth factor respectively. This is the first report of phosphorylation of tyrosine in a sequence related to a human hormone.