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A personal narrative is presented which explores the author's experience of working at the laboratory of Philip Thornber at the University of California-Los Angeles (UCLA) in the late 1970s.

Gly m Bd 28 K is a major soybean (Glycine maxMerr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the ß subunit of soybean ß-conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first ß-sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans. [ABSTRACT FROM AUTHOR]

The use of distributed (Internet) resources to enhance both traditional and distance education has caused much excitement in the science education community. However, one of the difficulties with relying on such freely available distributed resources has been the lack of certainty that the resources will be available for students next month, next semester, or next year. We have recently been involved in the development of three graduate-level biochemistry courses designed for high school teachers. Development of these courses relied heavily upon distributed science education resources. As a consequence, they represented a set of authentic science education resources that could be monitored over time to determine their rate of extinction. In total, the three courses contained 515 nonredundant URLs representing either scientific content of science education pedagogy. These have been monitored on a monthly basis during the 14 months since the creation of the courses (August 2000). During this period 85 (16.5%) of the URLs have ceased to function or had their content changed. The most attrition was seen in URLs with the “edu,” “com,” and “org” domain names, in which 17.5, 16.4, and 11% have already become inaccessible. [ABSTRACT FROM AUTHOR]

The activity of thylakoid protein kinase and the regulation of excitation energy distribution between photosystems I and II was examined during chloroplast biogenesis in light-grown Triticum aestivum (wheat) leaves. The specific activity of the thylakoid protein kinase decreased some six-fold during development from the young plastids at the base of the 7-d-old leaf to the mature chloroplasts at the leaf tip. Appreciable activity was also detected in plastids isolated from etiolated leaves. In mature chloroplasts the majority of phosphate was incorporated into the M=26,000 apo-proteins of the light-harvesting chlorophyll a/b-protein complex (LHCP). However, at early stages of chloroplast development and in the etioplast, the phosphate was predominantly incorporated into a polypeptide of M=9,000 dalton. Immature thylakoids, isolated from the base of the leaf, had relatively low concentrations of LHCP and could perform a State 1-State 2 transition, as demonstrated by ATP-induced quenching of photosystem II fluorescence. Analyses of photosystem I and photosystem II fluorescence-induction curves from intact leaf tissue demonstrated that this transition occurs in vivo at early stages of leaf development and, therefore, may play an important role in regulating energy transduction during chloroplast biogenesis. [ABSTRACT FROM AUTHOR]

Use of leaf meters to provide an instantaneous assessment of leaf chlorophyll has become common, but calibration of meter output into direct units of leaf chlorophyll concentration has been difficult and an understanding of the relationship between these two parameters has remained elusive. We examined the correlation of soybean ( Glycine max) and maize ( Zea mays L.) leaf chlorophyll concentration, as measured by organic extraction and spectrophotometric analysis, with output (M) of the Minolta SPAD-502 leaf chlorophyll meter. The relationship is non-linear and can be described by the equation chlorophyll (μmol m)=10(), r=0.94. Use of such an exponential equation is theoretically justified and forces a more appropriate fit to a limited data set than polynomial equations. The exact relationship will vary from meter to meter, but will be similar and can be readily determined by empirical methods. The ability to rapidly determine leaf chlorophyll concentrations by use of the calibration method reported herein should be useful in studies on photosynthesis and crop physiology. [ABSTRACT FROM AUTHOR]

Light-harvesting complex-II (LHC-II) phosphatase activity has generally been examined in the intact thylakoid membrane. A recent report of peptide-phosphatase activity associated with the chloroplast stromal fraction (Hammer, M.F. et al. (1995) Photosynth Res 44: 107-115) has led to the question of whether this activity is capable of dephosphorylating membrane-bound LHC-II. To this end, heat-treated thylakoid membranes were examined as a potential LHC-II phosphatase substrate. Following incubation of the thylakoid membrane at 60°C for 15 min, the endogenous protein phosphatase and kinase activities were almost eliminated. Heat-inactivated phosphomembranes exhibited minimal dephosphorylation of the light harvesting complex-II. Peptide-phosphatase activities isolated from the thylakoid and stromal fraction were able to dephosphorylate LHC-II in heat-inactivated phosphomembranes. The stromal phosphatase showed highest activity against LHC-II at pH 9. Dephosphorylation of the LHC-II by the stromal enzyme was not inhibited by molybdate, vanadate or tungstate ions, but was partially inhibited by EDTA and a synthetic phosphopeptide mimicking the LHC-II phosphorylation site. Thus, the previously identified stromal phosphatase does appear capable of dephosphorylating authentic LHC-II in vivo. [ABSTRACT FROM AUTHOR]

The study of the light-harvesting complex II (LHC-II) phosphatase activity has been difficult due to the membrane association of its substrate. Thylakoid membranes labeled with [γ-P]ATP were incubated with chymotrypsin, releasing phosphopeptides which served as labeled substrates for LHC-II phosphatase. Utilizing these phosphopeptides as substrates, protein phosphatase activities have been identified in both the thylakoid membrane and the stromal fraction. The thylakoid-bound phosphatase was liberated from the membrane with a sub-solubilizing concentration of Brij 35. The membrane and the stromal protein phosphatases were inhibited by NaF and EDTA, but not inhibited by microcystin-LR. The stromal phosphatase differed from the membrane phosphatase in pH optimum, in its lack of inhibition by molybdate ions, and by its response to magnesium and manganese ions. Using the soluble chymotryptic peptide substrate, the effect of light on pea thylakoid-bound LHC-II phosphatase activity was also assessed. Incubation of the thylakoid membranes in the light caused a 35% inhibition of LHC-II phosphatase activity. The inhibition was diminished by the addition of DCMU. Addition of 10 mM dithiothreitol stimulated the activity in darkness and obviated the inhibition when exposed to light. These studies suggest that positive or negative regulation of the LHC-II phosphatase activity is possible in vivo. [ABSTRACT FROM AUTHOR]

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25-27 kDa apoproteins of the light-harvesting chlorophyll a/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide at ca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light. [ABSTRACT FROM AUTHOR]

Aspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain. [ABSTRACT FROM AUTHOR]