Some observations on the ESCA spectra of plastocyanins.
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Biomedical subjects
Publications and source records attributed to D Boulter.
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A plant-algal type ferredoxin was isolated from the red alga, Porphyra umbilicalis. In its oxidised form the ferredoxin had absorption maxima at 277, (281), 323, 420 and 462 nm. Two atoms each of non-haem iron and labile sulphur were present per molecule protein. The midpoint potential of the protein was -400 mV and it effectively mediated electron transport in the NADP-photoreduction system of barley. The amino acid composition of Porphyra umbilicalis ferredoxin was determined as (Lys4, His2, Arg1, Asx10, Thr8, Ser7, Glx16-17, Pro3, Gly7, Ala8, Cys5, Val6, Met1, Ile5, Leu8, Tyr5, Phe2). The minimum molecular weight of approximately 11000 was confirmed by sedimentation-equilibrium studies in the analytical ultracentrifuge. Approaching half of the total amino acid sequence was determined by means of an automatic sequencer.
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We have measured the molecular weight of the small subunit of Fraction I protein from pea and broad bean by sodium dodecyl sulphate polyacrylamide gel electrophoresis, Sephadex gel-filtration and amino acid composition data. The results suggest a molecular weight of 12 000-14 500, although measurements by gel-filtration in alkali suggest a molecular weight of approximately 22 000. N-terminal amino acid sequence data and C-terminal determinations show that the protein consists of a single type of polypeptide chain, although the anomalously high molecular weight obtained on gel-filtration in alkali does not preclude the existence of the polypeptides as dimers under certain conditions.
The N-terminal amino acid sequence of plastocyanin from Stellaria media L. (chickweed) has been determined by means of an automatic sequencer. Amino acid derivatives obtained during the degradation were identified by a combination of t.l.c., g.l.c. and regeneration of the parent amino acid. The analytical results are presented for each sample, and the criteria used to establish the amino acid sequence are discussed.
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1. Meals were prepared from the seeds of fifteen varieties of cowpea (Vigna unguiculata), one of lima bean (Phaseolus lunatus) and one of yam bean (Sphenostylis stenocarpa), and their methionine content was determined by six different methods. 2. Total methionine content was determined by two chemical methods (ion-exchange chromatography and a colorimetric procedure) and by two microbiological methods. The 'available' methionine content was determined by microbiological assay with Streptococcus zymogenes. 3. All the different methods for total methionine determination gave similar results, with much the same high extent of precision. 4. The value for 'available' methionine content were similar to or marginally higher than the corresponding microbiological assay value for total methionine content. There was no indication that the methionine in any of the test samples was not completely available.
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The amino acid sequence of spinach (Spinacia oleracea L.) plastocyanin was determined. It consists of a single polypeptide chain of 99 residues and has a sequence molecular weight of 10415. The sequence was determined by using a Beckman 890C automatic sequencer and by the dansyl--phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. Overlap through the two methionine residues was not shown. Sedimentation equilibrium in the ultracentrifuge gave a molecular weight for spinach plastocyanin of about 9000, in contrast with the value of 21000 reported previously by Katoh et al. (1962).
The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either alpha-mannosidase or N-acetyl-beta-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.
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Proposed amino acid sequences of cytochrome c from nasturtium (Tropaeolum majus L.), box-elder (Acer negundo L.), elder (Sambucus nigra L.) and parsnip (Pastinaca sativa L.) are presented. Because of the very limited amounts of cytochrome available from some plant sources, peptides derived from the cytochromes c have been sequenced by the semi-quantitative dansyl-Edman technique (Gray & Hartley, 1963) without supporting quantitative amino acid analyses. Because of the qualitative nature of the work, the sequences proposed must be regarded as tentative. Considerations of homology, although useful as a guide, have been kept to a minimum in the construction of sequences. Only the nasturtium sequence relies on considerations of homology for a complete ordering of the peptides. Where material permitted, each residue of a proposed sequence was determined at least once from both a tryptic and a chymotryptic peptide.