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E S Kempner

Publications and source records attributed to E S Kempner.

At least 91 records · Page 5Linked to original sources
PubMed1982

Radiation inactivation of the glycoprotein, invertase.

The radiation target size for invertase activity has been determined for the Saccharomyces cerevisiae glycoprotein which contains 50% carbohydrate. Identical inactivation curves were observed for the native enzyme as well as samples depleted of carbohydrate by incubation with Endo-beta-N-acetylglucosaminidase H. The functional unit of 120,000 daltons was unaltered by the per cent of oligosaccharide cleaved by the enzyme, or by the presence or absence of the released sugars. The irradiated samples showed no change in hexose content even after radiation exposures which grossly destroyed enzymatic activity. Reducing sugars appeared in the irradiated samples, indicating radiation damage to the oligosaccharides. These results unequivocally identify the enzymatically functional portion of the invertase molecule as the polypeptide homodimer, independent of the covalently-bound carbohydrates, and indicate that transfer of radiation energy from protein to oligosaccharide or vice versa is inefficient.

Carbohydrates↗
PubMed1982

Radiation inactivation studies of the renal brush-border membrane phlorizin-binding protein.

Renal brush-border membrane vesicles were irradiated in the frozen state with a high energy electron beam. The integral membrane proteins, alkaline phosphatase and 5'-nucleotidase, each showed a single exponential loss of activity with radiation dose, indicating target sizes of 67,000 and 58,000 daltons, respectively. Inactivation of sodium-dependent phlorizin binding to the brush-border membrane D-glucose transporter was more complex. One-half of the phlorizin binding sites were lost after even the smallest doses of radiation suggestive of large functional units (greater than 4 X 10(6) daltons) for a subpopulation of phlorizin binding proteins. The remaining sites behaved as a single radiation target of 110,000 +/- 8,000 daltons and retained the kinetic characteristics commonly associated with phlorizin binding to the glucose transporter. Thus, the data are consistent with the assignment of a molecular weight of 110,000 to the phlorizin binding moiety of the brush-border membrane D-glucose transport protein.

5'-Nucleotidase↗
PubMed1982

The functional unit of the arom conjugate in Neurospora.

The functional unit of arom polyenzyme conjugate of Neurospora crassa was determined by analysis of radiation inactivation of each of the five activities in the conjugate. The functional targets for all five enzymes were in close agreement with the value of 300,000 obtained by conventional hydrodynamic procedure for the native dimeric structure. These data indicate that at least 95% of the functional enzyme system in crude extracts exists in a dimeric form and that both polypeptide chains of the homodimer are required for full activity of each of the five enzymes.

Alcohol Oxidoreductases↗
PubMed1982

The size of the thyroid hormone receptor in chromatin.

We have used radiation inactivation and target theory to determine the size of the functional unit for T3 binding in rat liver chromatin. The process involves exposure of frozen chromatin samples to a beam of high energy electrons produced in a linear accelerator and subsequent measurement of the residual capacity to bind hormone. Our experiments were carried out using three forms of solubilized chromatin: 1) sonicated, containing the receptor in fragments which sedimented faster than 30 S; 2) digested by nuclease, containing the receptor in a form which sedimented at 5-6 S; 3) digested by nuclease and made 0.5 M in KCl, containing the receptor in a form which sedimented at 3.8 S. We have shown that in each sample preparation the receptor retained the ability to bind T3 with the same capacity and affinity that had previously been measured with high molecular weight chromatin. Irradiation caused a reduction in the capacity to bind T3 but did not change the affinity of the remaining receptors for the hormone. In each preparation, the radiation resulted in a simple exponential loss of binding capacity with dose, indicating that a single target size was detected. Within the variation of the measurements, the target size for each form of the receptor was the same, 59,000 daltons.

Animals↗
PubMed1981

Demonstration by radiation inactivation that insulin alters the structure of the insulin receptor in rat liver membranes.

Using the technique of radiation inactivation we have previously shown that the insulin receptor in rat liver membranes is composed of at least two functional components: an insulin binding component and a component which acts as an affinity regulator ((1980) J. Biol. Chem. 255, 3412-3419). In the present study, we have used this technique to examine whether insulin alters the size or structure of the insulin receptor. When insulin is bound to its receptor in liver membranes before radiation, there is a marked change in the inactivation profile. Analysis of the results of indicated that insulin binding to its receptor caused a decrease in the functional size of the insulin binding component from 115,000 to 76,000 and also diminished the interaction between the binding and regulatory components. The magnitude of these effects depended on the insulin concentration, and analogues of insulin with reduced receptor affinity show a reduced effect. Under these conditions, there was no change in the apparent size of the affinity regulator (about 260,000). These data suggest that the interaction of insulin with its receptor in situ produces a change in receptor structure which may be important in signal transmission.

Animals↗
PubMed1981

Molecular size of the hepatic receptor for asialoglycoproteins determined in situ by radiation inactivation.

Radiation inactivation was used to determine the functional molecular size of the rat liver membrane protein which binds desialylated glycoproteins. Purified plasma membranes from rat liver were irradiated with high energy electrons from a linear accelerator and then assayed for 125I-asialo-orosomucoid binding activity. Target size analysis of the data revealed that increasing doses of ionizing radiation from 1-48 megarads resulted in a monoexponential decay in binding activity due to a decrease in the number of available binding sites; dissociation and binding affinity were unaffected. The molecular weight of the rat binding protein, determined in situ by target analysis, was 104,000 +/- 17,000; that of the rabbit binding protein was 109,000 +/- 5,000. Comparison of the value obtained by irradiation of the intact rat plasma membrane with that of the purified receptor revealed the latter to have an apparent molecular weight of 148,000 +/- 16,000. Evidence is presented to indicate that the observed increase in target size was a response to the presence of Triton X-100 used in the solubilization and assay procedure. In contrast to the size of the ligand binding functional unit, the antireceptor antibody binding site was estimated to be 30,000 +/- 2,000.

Animals↗
PubMed1981

Functional unit of the low density lipoprotein receptor of fibroblasts: a 100,000-dalton structure with multiple binding sites.

The low density lipoprotein (apoprotein B,E) receptors of fibroblasts bind plasma lipoproteins that contain either the B (apo-B) or E (apo-E) apoproteins. These include the low density lipoproteins (LDL) containing apo-B and certain high density lipoproteins containing apo-E (e.g., the cholesterol-induced apo-E HDLc). The same receptor binds both LDL and apo-E HDLc, but the apo-E HDLc bind with much higher affinity. This higher affinity is due to the binding of apo-E HDLc to multiple receptor sites. One possible structural model for this receptor is that each apo-B,E receptor possesses a single binding site. Thus, the multiple binding of apo-E HDLc would require the recruitment of four independent receptors to bind to a single apo-E HDLc particle. A second model is that each receptor unit possesses multiple binding sites capable of binding one apo-E HDLc particle or four LDL particles. This study characterizes the apo-B,E receptors in situ in the membranes of fibroblasts by radiation inactivation. This technique allows one to determine the functional size of the receptor on the basis of the amount of radiation required to inactivate the structure. The larger the molecular size of the structure, the less ionizing radiation (generated by a linear accelerator) that will be required to abolish the receptor--ligand interaction. The functional size of the apo-B,E receptor is 106,000 Mr as determined with both LDL and apo-E HDLc binding after radiation inactivation. Furthermore, the data derived from radiation inactivation and Scatchard analysis indicate that each apo-B,E receptor has multiple binding sites and that each functional receptor unit, capable of binding one apo-E HDLc or four LDL particles, has a molecular weight of 106,000.

Apolipoproteins↗
PubMed1980

The functional unit of polyenzymes. Determination by radiation inactivation.

Recently, target analysis has been re-evaluated as a technique for the determination of molecular sizes (Kempner, E. S. & Schlegel, W. (1979) Anal. Biochem. 92, 2-10). The technique yields the size of the functional unit, i.e. the minimal assembly of structures necessary for a given function such as an enzymatic activity. Using this method, we have not determined the sizes of the functional units for different enzymatic activities on the "arom" conjugate from Euglena, a polyenzyme catalyzing five sequential reactions in the shikimic acid pathway, and on two conjugates from Escherichia coli carrying both aspartokinase and homoserine dehydrogenase activities. In each conjugate, the size for different enzymatic activities was measured and found to be the same. When compared to the molecular weight obtained with other techniques, the target size matched either the entire conjugate (aspartokinase-homoserine dehydrogenase conjugates I and II) or half the unit ("arom" conjugate). The information was obtained with minimal perturbation of the complexes and sparing laborious purification and reconstitution experiments. Tryptophan synthase was irradiated both as an intact conjugate and also as isolated subunits. In both structural forms, beta 2 was identified as the functional unit for the conversion of indole and serine to tryptophan. The results of this study give insight into the structural assembly of these polyenzymes.

Animals↗
PubMed1979

Synchronization of division in vitamin B12-starved Euglena gracilis.

Vitamin B12 deficiency arrests cell division in Euglena gracilis. B12 starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B12 deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.

Animals↗
PubMed1979

Absolute DNA determinations by flow microfluorometry.

The DNA content of Euglena gracilis and murine leukemic cell line L-1210 were varied by growth under abnormal conditions. DNA measurements by colormetry and by flow microfluorometry were compared and absolute calibration of the flow device was established.

Animals↗