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B Zeeberg

Publications and source records attributed to B Zeeberg.

26 records · Page 2Linked to original sources
PubMed1979

Determination of free and bound microtubular protein and guanine nucleotide under equilibrium conditions.

The dissociation constant for GDP binding to the E site of tubulin isolated by chromatography on Sepharose 6B is equal to 6.1 X 10(-8) M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value, and the discrepancy with earlir results is analyzed. By use of a recently described column centrifugation procedure [Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899], it was established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP, and the dissociation constant is 2.2 X 10(-8) M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. This has been used to determine that the dissociation constant for GDP bound to tubulin is equal to 5.5 X 10(-8) M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment [Dixon, H. B. F. (1976) Biochem. J. 159, 161-162] of ligand loss during exclusion chromatography is discussed.

Animals↗
PubMed1978

Reactions of tubulin-associated guanine nucleotides.

Only exchangeably bound nucleotide (E-site) is involved in the reaction of the transplhosphorylase activity in microtubular protein. Contrary to earlier reports, we find that the nonexchangeable nucleotide (N-site) is not a substrate. This conclusion is based upon comparison of: (a) rates of hydrolysis of endogenous tubulin-associated GTP and added [32p]GTP: (b) hydrolysis rates for added [32p]GTP and [3h]GTP; (c) the 32P/3H ratio in bound and free GTP after reaction with [3h, 32p]gTP. During the course of the above studies we have made the unusual observation of a time dependent augmentation in the expected amount of GTP relative to GDP at the E-site; there is either a net conversion of E-site GDP to E-site GTP, or a means for providing additional E-site GTP from another source.

Glycoproteins↗
PubMed1977

Microtubular protein catalytic interactions with nucleotides.

Purified tubulin prepared from pig brain in the absence of added guanine nucleotides contains 1 mol each of GDP and GTP/mol of tubulin. Incubation of a tubulin preparation with inorganic [32P]phosphate results in the incorporation of 32P into tubulin-associated GDP and GTP. Typically, in a 5-h incubation, 0.45 mM 32Pi reacts with 22 muM tubulin to form 3.3 muM [32P]GDP, and 0.3 muM [32P]GTP. The yield of labeled nucleotide is decreased as the result of a hydrolase activity associated with the preparation. The [32P]GTP is exclusively beta-labeled and is therefore formed by phosphorylation of [32P]GDP by a phosphate donor other than inorganic phosphate, most likely by nonradioactive GTP. Added GDP significantly decreases the yield of labeled GTP, but increases the yield of labeled GDP in a manner that is consistent with a partial protection against [32P]GDP hydrolysis, but not consistent with significant additional [32P]GDP formation. Added GMP is an inhibitor of both labeling activities associated with the preparation, although it has no effect on the hydrolase activity. Added ADP (0.4 MM) does not form labeled ADP or ATP and does not influence the labeling of GDP or GTP. The formation of [32P]GDP was shown to occur by an exchange mechanism rather than through net synthesis from GMP and Pi. These results provide evidence for a reversible guanidylation of the protein. The labeling activity is always specifically associated with highly purified tubulin preparations. Microtubular protein preparations are found to catalyze an exchange of oxygen from H218O into inorganic phosphate. Thus, there are two distinct catalytic properties associated with tubulin.

Animals↗
PubMedDate not supplied

Chlamydia trachomatis antigen detection by Chlamydiazyme combined with Chlamydia Blocking Reagent verification.

Several options exist for the detection of chlamydial infection in a routine laboratory setting. Enzyme immuno assay (EIA) technology offers rapid turn around of results and is less technically demanding than chlamydial cell culture. In addition, recently introduced EIA confirmatory reagents have the potential to improve the accuracy of EIA detection. We have evaluated one such confirmatory reagent (Chlamydia Blocking Reagent, Abbott Laboratories) to determine the accuracy of the Chlamydiazyme EIA with special regard to interpretation of low absorbance values. An initial series of 192 male urethral specimens showed that use of a lowered cut off level (absorbance value 0.05) compared with that recommended by the manufacturer increased sensitivity of the EIA from 0.73 to 0.83, thus motivating studies on this interpretative modification. Of 1101 EIA reactive specimens, 65% were determined to be chlamydia positive by the Chlamydia Blocking Reagent. The proportion of female cervical specimens that did not confirm positive was elevated compared with male urethral specimens, 43% vs. 5.7% respectively. In samples yielding absorbance from the recommended cut off level to 0.05 (approximately 50% below), the corresponding figures were 78% and 14% respectively. In 85 selected EIA reactive samples, examination by a direct immunofluorescence staining assay (DFA) (MicroTrak, Syva Inc.) revealed elementary bodies in 85% of 67 blocking test positive and in 24% of 18 blocking test negative samples. The possibility that Gram-negative bacteria were responsible for unconfirmed EIA reactive specimens was investigated using bacterial suspensions. While EIA reactivity was noted with several strains for Gram-negative bacteria, both the blocking reagent and DFA correctly verified the absence of chlamydial antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Bacterial↗