The binding of biotin to tetrameric avidin changes the environment of tryptophan residues. Binding reduces the total tryptophan fluorescence by 34%, shifts the emission peak from 337 to 324 nm, and reduces the fluorescence bandwidth from 61 to 46 nm. These changes are consistent with the movement of tryptophans to a nonpolar, internal environment. In the absence of biotin, iodide readily quenches the fluorescence of 20-29% of the initial fluorescence, which likely corresponds to one tryptophan located in a positively charged environment. Iodide may have weak access to additional fluorescence, corresponding to perhaps one additional tryptophan. Acrylamide, in the absence of biotin, has good access to three-fourths or more of the fluorescence, but the remainder, due to one or two tryptophans, is well shielded. The binding of biotin completely prevents iodide quenching and decreases acrylamide access dramatically. The data indicate that biotin binding shifts two or three tryptophans to an internal, hydrophobic, shielded environment.

Acrylamides↗

PubMed1989

Postsecretory modifications of streptavidin.

Streptavidin, an extracellular biotin-binding protein from Streptomyces avidinii, exhibits a multiplicity in its electrophoretic mobility pattern which depends both upon the conditions for growth of the bacterium and upon the protocol used in the purification of the protein. The observed structural heterogeneity appears to reflect the action of two types of postsecretory molecular events: proteolytic digestion of the intact Mr-18,000 subunit to a minimal molecular size (approx. Mr 14,000), and aggregation of the native tetramer into higher-order oligomeric forms. The extent of subunit degradation and/or tetrameric aggregation affects the capacity of a given streptavidin preparation to interact with biotin-conjugated proteins in different assay systems.

Amino Acid Sequence↗

PubMed1989

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.

Avidin↗

PubMed1989

Blood levels and serum protein binding of cis-platinum(II) complexed to carboxymethyl-dextran.

Plasma levels and serum protein binding of cis-diamminedichloroplatinum(II) (cis-DDP) or cis-diamminediaquoplatinum(II) (cis-aq) complexed to carboxymethyl-dextran (CM-dex) with a molecular weight of 10,000 (T-10), 40,000 (T-40), and 250,000 (T-250) were investigated in BALB/c mice. Levels of active drug in the circulation after the i.v. or i.p. administration of the free or complexed drugs, as well as the loss of drug activity due to serum protein binding following incubation with mouse serum, were monitored by an antitumor in vitro assay using a drug-sensitive tumor cell line. Following i.v. injection of the complexes, active platinum(II) was maintained in the circulation at higher levels and for a longer period, whereas the free drug disappeared rapidly. The rate of disappearance of the complexed drug from the circulation was markedly influenced by the molecular size of the carrier CM-dex, since the retained amount of drug was considerably higher with the T-40 and T-250 complexes than with the T-10 complex. An i.p. injection resulted in a rapid and transient appearance of low levels of the free drugs in the blood, whereas in the case of the complexes, transport to the circulation was slower and their maintenance in the blood system was markedly higher. Serum protein binding was much slower with CM-dex-complexed drugs (regardless of the molecular size of the CM-dex carrier) than with the free drugs.

Animals↗

PubMed1989

A thiophilic adsorbent for the one-step high-performance liquid chromatography purification of monoclonal antibodies.

Thiophilic adsorption chromatography, developed originally by Porath and colleagues (1985, FEBS Lett. 185, 306-310) for conventional chromatographic techniques, was transformed to the HPLC mode upon preparation of new and improved sulfur-containing silica beads. The new thiophilic adsorbent is of high capacity and is suitable for the rapid and single-step purification of all subclasses of monoclonal and polyclonal antibodies. Due to its broad specificity, the thiophilic silica column is an efficient, stable, and inexpensive substitute for protein A and protein G columns used today to purify antibodies.

Adsorption↗

PubMed1989

Localization of serotonin receptors in the rat thalamus by electrophysiology and the action of 5-HTP-DP-hex.

This study was designed to pinpoint the site at which N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP-hex) exerts its previously reported effect on thalamic neurons in rats. The animals were prepared under chloralose-urethane anesthesia for a stereotaxic approach to either the nucleus ventralis posterolateralis (nVPL) or the centrum medianum-parafascicularis complex (CM-Pf) of the thalamus. Individual neurons in these nuclei were separately activated by single-pulse stimulation of the sciatic nerve or the thalamic fibers that form reciprocal connections between the CM-Pf and the second somatosensory (SII) region of the nVPL. Poststimulus time histograms were constructed from computer readouts of the stimulus-evoked responses of a neuron during a 500-ms period accumulated in a digital computer 100X. In addition, the number of spikes accumulated in each histogram was compared to the number of spikes accumulated under identical conditions on the same neuron after intracarotid infusion of 5-HTP-DP-hex. The effect of the drug was reversed by the infusion of 5-HTP. Statistical evaluation of the accumulated spike counts indicated that 5-HTP-DP-hex suppressed only the excitation of CM-Pf neurons from the SII of the nVPL; the input of the sciatic nerve into the CM-Pf remained unaltered. Furthermore, no effect was exerted by this dipeptide on the afferent excitation of neurons in the SII of the nVPL.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptophan↗

PubMed1989

Avidin-biotin technology ten years on: has it lived up to its expectations?

Following the practical success attributed to avidin-biotin technology, researchers are now returning to the basics to try to understand the structural and functional requirements for the high-affinity avidin-biotin interaction.

Amino Acid Sequence↗

PubMed1989

Effects of covalently bound silica-nitroimidazole drug particles on Entamoeba histolytica.

Most commonly used antiamoebic drugs are effective in invasive amebiasis, but their response against trophozoites of Entamoeba histolytica, present in the lumen of the human colon, is inadequate. We report the development of an antiamoebic drug carrier that may be effective against luminal infections. Our preparation consists of small silica particles (5-10 microns in diameter) covalently linked to a potent antiamoebic drug, 2-(4-aminophenoxymethyl)-5-nitro-1-methyl imidazole. Silica-drug particles were injected into mice, hamsters, and guinea pigs. We found that trophozoites phagocytosed the particles in vivo and in vitro, followed by rapid cell death due to the released drug. Analysis of mouse serum revealed that no drug was absorbed from the intestine after placement of the drug-containing particles in the intestine. The antiamoebic activity of particles recovered from the intestine was almost fully retained. This novel antiamoebic concept may be useful for luminal therapy for asymptomatic amebiasis and may minimize side effects and frequency of administration.

Amebicides↗

PubMed1988

Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle.

We report here on the purification and characterization of a new 25-kDa inhibitor of actin polymerization from turkey gizzard smooth muscle. The protein was purified by chromatography on DEAE-cellulose and hydroxyapatite, as well as by affinity chromatography on an immobilized-antibody column. The purified polypeptide reduced the low-shear viscosity of actin, apparently due to its inhibitory effect on actin polymerization. We demonstrate that this protein is largely responsible for the apparent inhibitory activity previously reported to be associated with smooth muscle vinculin preparations. Three independent monoclonal antibodies prepared against the 25-kDa inhibitor of actin polymerization can effectively adsorb the inhibiting activity of actin polymerization from the crude vinculin preparation or inhibit it. We also show here that the 25-kDa inhibitor of actin polymerization tends to undergo dimerization when maintained in non-reducing buffers, concomitant with the loss of its inhibitory activity. Immunohistochemical labeling of frozen sections, as well as immunoblotting analyzes, indicated that the 25-kDa inhibitor of actin polymerization is particularly enriched in smooth muscle cells and that its distribution is apparently homogenous throughout the cytoplasm showing no apparent enrichment in the vinculin-rich dense plaques located along the endofacial surface of the plasma membrane.

Actins↗

PubMed1988

Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site.

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

Amino Acid Sequence↗

PubMed1988

p-Diazobenzoyl-biocytin: a new biotinylating reagent for DNA.

Explore the source record for details and available documents.

Biotin↗