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M L Pratt

Publications and source records attributed to M L Pratt.

10 recordsLinked to original sources
PubMed1988

Neurospora tryptophan synthase. Characterization of the pyridoxal phosphate binding site.

Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM. The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli. The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride. The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate. A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced. The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E. coli enzymes.

Amino Acid Sequence↗
PubMed1983

Heterogeneity of binding sites for the pyruvate dehydrogenase component on the dihydrolipoyl transacetylase core of bovine kidney pyruvate dehydrogenase complex.

We have characterized the dissociation equilibrium constant (Kd) and the rate constants of association and dissociation for the binding of the pyruvate dehydrogenase component (PDH) to the dihydrolipoyl transacetylase component of kidney pyruvate dehydrogenase complex. We have found about 7 high-affinity sites (Kd = 1.5 X 10(-11) M) and about 13 low-affinity sites (Kd = 2.5 X 10(-8) M) with negative cooperativity for binding of PDH at the weaker sites. The high-affinity sites show a much higher rate constant for association of PDH with the core than do the low-affinity sites. Catalytic turnover strengthens binding of PDH at high-affinity sites when the complex is preincubated in the presence of thiamin pyrophosphate (TPP). In the absence of TPP, appreciably tighter binding occurs at the low-affinity sites (Kd = 5.9 X 10(-10) M). TPP weakens PDH binding at these sites with a half-maximal effect at about 5 microM TPP. In addition to TPP, increased ionic strength or 1-3 mM Mg2+ (with an enhanced effect of Mg2+ at higher ionic strengths) weakens PDH binding at low-affinity sites with a corresponding increase in the rate constant for dissociation. Further studies will be required to determine whether site heterogeneity contributes to dynamic processes in the function and regulation of the pyruvate dehydrogenase complex.

Acetyltransferases↗
PubMed1982

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: preparation and characterization.

The dihydrolipoyl transacetylase core components of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N,N'-p-phenylenedimaleimide. In one approach, the N,N-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found that N,N-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues. Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.

Acetyltransferases↗
PubMed1982

Purification of bovine kidney and heart pyruvate dehydrogenase phosphatase on Sepharose derivatized with the pyruvate dehydrogenase complex.

Pyruvate dehydrogenase phosphatase has been purified to apparent homogeneity from mitochondrial extracts of both beef heart and beef kidney. An essential step in this three-step purification is affinity chromatography of a largely purified phosphatase fraction using Sepharose beads to which pyruvate dehydrogenase complex is covalently bound through the lipoic acid residues of the dihydrolipoyl transacetylase component of the complex. The purified phosphatase, which has a native relative molecular mass, Mr, of about 140000, is composed of two nonidentical subunits of Mr 89000 and 49000.

Animals↗
PubMed1979

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.

Biological Transport↗
PubMedDate not supplied

Computer simulation analysis of blood donor queueing problems.

A computer simulation model was used to study work flow and queueing problems in blood collection. Donor scheduling strategies, such as stream and block arrivals, were compared with random arrival at a constant hourly rate. Controlling donor queueing required keeping donor arrivals from exceeding the system capacity, rather than using any one type of scheduling strategy. However, widely uneven arrivals compared to even arrivals at the same input level caused protracted queueing. Because men have a shorter average phlebotomy processing time, time can be saved in some situations by scheduling mostly men at the beginning of a drive. Nursing utilization appears optimal at donor arrival rates at or near system capacity. Techniques may be available to modify the collection system to ameliorate or prevent queueing when donor arrivals appear to be excessive.

Blood Donors↗