Direct fluorimetric assay for proteolytic activity against intact proteins.
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Anilino Naphthalenesulfonates↗
Biomedical subjects
P W Spencer
Publications and source records attributed to P W Spencer.
The occurrence and nature of ornithine carbamoyltransferase in senescing apple leaf tissue.
Ornithine carbamoyltransferase (EC 2.1.3.3) activity was detected in apple (Pyrus malus L.) leaf tissue from early June to November. Total activity remained relatively constant at 4.1 mumoles citrulline produced per hour per 10 cm(2) until mid-October when it sharply doubled. Following the first frost of the autumn, the enzyme lost about 80% of its former activity. The enzyme from apple leaf exhibited two pH optima, one at pH 8.6 and the other at pH 7.8, indicating the presence of isozymes or two forms of the enzyme. At pH 8.6, a partially-purified enzyme preparation had binding contrasts for its substrates of 6 mm for carbamyl-phosphate and 4.8 mm for ornithine. At pH 7.8, the Km for carbamyl-phosphate was 1.9 mm and the Km for ornithine was 1.22 mm.
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Globulin-specific Proteolytic Activity in Germinating Pumpkin Seeds as Detected by a Fluorescence Assay Method.
The proteolytic activities of alpha-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds (Cucurbita moschata) were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates. Chymotrypsin, trypsin, pepsin, and bromelain exhibited activity against all or almost all of the protein substrates. The activity of 1 mug of alpha-chymotrypsin or trypsin and 100 ng of pepsin could be easily detected by this method of assay within 4 to 5 minutes depending upon the substrate. The enzyme extracted from 3-day germinated pumpkin seeds exhibited strong activity only against pumpkin seed globulin, weak activity against the globulins of squash and cucumber and casein, and no activity against the other protein substrates. Activity against pumpkin globulin was maximal at pH 7.4. When assayed by an increase in ninhydrin-positive products, the enzyme extract from pumpkin seeds also showed strong activity against pumpkin globulin and weak activity against casein. The 1-anilino-8-naphthalenesulfonate-fluorescence method was at least 20 times more sensitive than the ninhydrin method and was 10 to 20 times more rapid.
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Apple leaf senescence: leaf disc compared to attached leaf.
Attached apple leaves (Pyrus malus L., Golden Delicious) began to lose protein in early August as the first sign of senescence. Apple leaf discs prepared from samples before early August gained protein for up to 7 days after detachment. After early August, the loss of protein from leaf discs was no greater than the loss from attached leaves in 7 days. The loss of chlorophyll from leaf discs began over 2 months before attached leaves began to lose chlorophyll naturally and before leaf discs lost protein. Leaf discs from presenescent leaves did not senesce significantly faster when maintained in darkness instead of 12 hours of light. In general, the loss of protein and chlorophyll from apple leaf discs after 7 days was much less than for most other leaf types studied.
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Incorporation of C-Leucine into Apple Leaf Protein and Its Inhibition by Protein Synthesis Inhibitors during Growth and Senescence.
Of the total (14)C-leucine taken up by intact apple (Pyrus malus L., Golden Delicious) leaf discs, 44 to 62% is incorporated into protein from June to early October. Of this amount, an average of 35% is released by mild, room temperature acid hydrolysis. Prior to mid-August when leaf protein begins to decline, 15 to 20% of the (14)C-leucine incorporated into protein occurs in water-(buffer) soluble protein, of which only 3% is released by mild acid hydrolysis. After mid-August, 40% of the label in protein occurs in soluble protein. The specific radio-activity of the soluble protein increases by 4- to 5-fold after mid-August, while that of total protein increases by less than 2-fold. In presenescent leaves (before the decline of protein in August) 20 micrograms per milliliter cycloheximide inhibits the incorporation of (14)C-leucine into protein by 71%, and 20 micrograms per milliliter chloramphenicol inhibits it by 30%. In senescing leaves, cycloheximide inhibits (14)C-leucine by 85% or more, while chloramphenicol inhibits it by less than 15%. Coincident to the initial decline of leaf protein, chloramphenicol greatly loses its ability to inhibit the incorporation of (14)C-leucine into apple leaf protein. At all leaf ages, chloramphenicol increases the loss of chlorophyll from apple leaf discs. The effect of cycloheximide on leaf disc senescence changes with leaf age: in early season samples, it increases the loss of chlorophyll; in mid-season samples, it has no effect; and in late season samples, it retards the loss of chlorophyll.
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Biochemical and Enzymatic Changes in Apple Leaf Tissue during Autumnal Senescence.
The biochemical changes occurring during the natural senescence of apple leaf tissue (Pyrus malus L., Golden Delicious) coincided with specific changes in the environment. Protein, sugars, and total nitrogen began declining in leaf tissue when the daylength first became less than 14 hours in the second week of August. The activity of triose phosphate dehydrogenase declined shortly afterwards, while the activities of malate dehydrogenase, glutamic dehydrogenase, and aspartate aminotransaminase increased. Chlorophyll, DNA, RNA, and fresh weight began declining when the daylength first became less than 12 hours at the end of September. At the same time sugars and the activities of RNase, polyphenol oxidase, and proteolytic enzymes began increasing. Protein synthesis, total nitrogen, and the activities of malate dehydrogenase, glutamic dehydrogenase, and aspartate aminotransaminase began declining rapidly and amino acids began to accumulate after the first frost of the year. RNase, polyphenol oxidase, and proteolytic activity reached their highest specific activities after the first frost.
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