Separation of labeled from unlabeled proteins by equilibrium density gradient sedimentation

Hu, Ankang; Bock, Robert M.; Halvorson, Harlyn O.

doi:10.1016/0003-2697(62)90129-x

Cited by 117 publications

(25 citation statements)

References 9 publications

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“…* The enzyme's molecular weight is ca 41,000 and it appears to be a monomer. Previous evidence has shown that the smaller the protein, the broader the peak after isopycnic centrifugation (16). Fig.…”

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confidence: 83%

“…The procedure of Chrispeels and Varner (15) (15)(16)(17). An initial experiment with pure catabolic dehydroquinase using the method of Chrispeels and Varner (15) revealed that the native enzyme had a density of 1.2790 g/cm3.…”

mentioning

confidence: 99%

“…Density labeling with D20 was a novel approach to the problem and had never been tried previously with N. crassa to our knowledge. The technique of density labeling followed by isopycnic equilibrium centrifugation was originally utilized with Escherwchia coli and subsequently applied to studies of protein synthesis in germinating seeds (15)(16)(17). Studies of this type using catabolic dehydroquinase proved quite definitive because this high-molecularweight enzyme gave a sharp activity peak after centrifugation.…”

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confidence: 99%

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Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Reinert

Giles

1977

Proc. Natl. Acad. Sci. U.S.A.

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In Neurospora crassa three inducible enzymes are necessary to catabolize quinic acid to protocatechuic acid. The three genes encoding these enzymes are tightly linked on chromosome VII near methionine-7 (me-7). This qa cluster includes a fourth gene, qa-1, which encodes a regulatory protein apparently exerting positive control over transcription of the other three qa genes. However, an alternative hypothesis is that the qa-I protein simply activates preformed polypeptides derived from the three structural genes. The use of density labeling with D20 demonstrated conclusively that the qa enzymes are synthesized de novo only during induction on quinic acid. Native catabolic dehydroquinase (5-dehydroquinate dehydratase; 5-dehydroquinate hydro-lyase, EC 4.2.1.10) (a homopolymer of ca 22 identical subunits) has a density of 1.2790 g/cm3 as determined by centrifugation in a modified cesium chloride density gradient. Growth in H20 followed by induction in 95% D2O shifts the density of the enzyme to 1.3130 g/cm3, indicating de novo synthesis during induction. In the reciprocal experiment, i.e., growth in 80% D20 followed by induction in either 95% D20 or H20, the densities of catabolic dehydroquinase were 1.3135 and 1.2800 g/cm3, respectively. Because growth on D20 does not affect the density of the H20-induced enzyme, there can be no significant synthesis of catabolic dehydroquinase prior to induction. Similar results were obtained for a second qa enzyme, quinate dehydrogenase (quinate:NAD+ oxidoreductase, EC 1.1.1.24). Thus, induction of two qa enzymes involves de nQvo protein synthesis, not enzyme activation or assembly. In Neurospora crassa, the catabolism of quinic acid to acetate for use as a carbon source has been amply studied (1-3). Recent interest has centered on the initial three inducible catabolic enzymes necessary to convert quinic acid to protocatechuic acid (4-13). These enzymes are not physically associated with one another, yet the three genes encoding them are tightly linked on chromosome VII near me-7. In Giles, unpublished). This qa gene cluster also includes a fourth gene, qa-1, which apparently regulates the transcription of the three other genes in the cluster.There is strong genetic evidence that the qa-1 gene encodes a regulatory protein which, in the presence of quinic acid, exerts positive control over the synthesis of the enzymes encoded in the three adjacent structural genes (6, 10). The appearance of enzyme activity is very specifically regulated. In the presence of a preferred carbon source, e.g., sucrose, the activities of all three enzymes are quite low. Quinate as a sole carbon source causes a coordinate induction of all three enzymes (9). This increase in enzyme activity can be inhibited by the simultaneous addition of cycloheximide to the medium (ref. 5; J. A.Hautala, unpublished data; this paper). This effect strongly suggests that all three enzymes are being synthesized de novo during induction. An alternate explanation, which as yet has never been conclusively eliminate...

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Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Reinert

Giles

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…The most convincing method to show that an enzyme is'synithesized de novo is the densi,ty labeling technique (2) employing equilibrium density gradient sedimentation (12). Seeds were germinated in D.2O to density label newly synthesized proteins.…”

Section: C-2-uiridinementioning

confidence: 99%

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Gientka-Rychter¹,

Cherry²

1968

Plant Physiol.

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Receiv-ed NovTember 20, 1967. A bstract. Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 tg/ml) during water imbibition inhibited the increase in isocitritase aotivity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA syn-thesis was inhibited. Germination of peanut seed in 14C-reconstituted amino acids followed by fractionation of a 20 to 35 %c ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45 %o D^O followed by enzyme purification and CsCI equilibrium cenitrifugation revealed that all the enzyme from D,O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.

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“…Do the rates of synthesis and degradation of the two gene products differ from one another? We have attempted to answer these questions by the techniques of starch gel electrophoresis (2) and density labeling (3,4 V4. The polypeptide product of the Ct2 locus is tentatively designated subunit Z; the homotetramer is Z4.…”

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Turnover of Genetically Defined Catalase Isozymes in Maize

Quail

Scandalios

1971

Proc. Natl. Acad. Sci. U.S.A.

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The turnover of two homotetramers of catalase, V4 and Z4, specified by genes at two separate loci, has been studied during germination of maize seed by the techniques of density labeling and starch-gel electrophoresis. Both isozymes were shown to be turning over during this time. However, Z4 accumulates because the rate of synthesis exceeds the rate of degradation, whereas V4 slowly disappears because the rate of degradation exceeds the rate of synthesis. Evidence is also presented that the rate of synthesis of Z4 exceeds that of V4, which suggests that this may be a major factor in the differential expression of the two catalase genes.Maize catalase (H202:H202 oxidoreductase, EC 1.11.1.6) is a tetramer (1). The products of catalase genes at two distinct loci, Ct1 and Ct2, have been detected and shown to be inherited independently of each other (Scandalios, manuscript in preparation). When present together at the same stage of development, the two gene products (subunits) interact to form active tetramers. However, from the time of pollination to just prior to seed maturation, only the product of the Ct1 locus is present in the developing caryopsis of inbred lines homozygous at this locus. Thus, only one isozyme (the homotetramer) is active during this time. The product of the Ct2 locus is first detected in the dry seed and increases dramatically during the first 3 days of germination. The Ct1 gene product, on the other hand, slowly decreases in activity during germination and ultimately disappears. During the time that both classes of subunits are available, five species of active tetramers are detected-two homotetramers and three heterotetramers. Mich. 48823 V4. The polypeptide product of the Ct2 locus is tentatively designated subunit Z; the homotetramer is Z4. In this inbred line, the Z4 isozyme has a greater electrophoretic mobility than the V4 isozyme. MATERIALS AND METHODS Growth and extraction of seedlingsSeeds of maize, inbred line W64A, were germinated between moistened filter papers in Petri dishes in the dark at 240C.The papers were moistened either with 10 mM K'4NO3 in H20 (14N-H20) or with 10 mM (99 atom %) K15N03 in 70% D20 ('5N-D20). In the chase experiments, seeds were germinated for 36 hr in 15N-D20, rinsed for 20 min with several changes of water, and then placed in '4N-H20 for the chase period. Twenty scutella from each treatment were homogenized with sand in a mortar and pestle in 1 ml of 25 mM glycylglycine buffer (pH 7.4) and centrifuged at 20,000 X g for 10 min. The crude supernatant was used directly for electrophoresis. ElectrophoresisStarch-gel electrophoresis and staining for catalase were performed as described (2). For preparative purposes the entire extract from twenty scutella was applied to a single starch gel. After electrophoresis, a thin slice of the gel was stained to facilitate location of the V4 and Z4 isozymes. Strips corresponding to these two isozymes were cut from the remainder of the gel and centrifuged at 30,000 rpm for 30 min in an International B60 ultra...

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Separation of labeled from unlabeled proteins by equilibrium density gradient sedimentation

Cited by 117 publications

References 9 publications

Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Turnover of Genetically Defined Catalase Isozymes in Maize

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