Analysis of heterogeneity in nonspecific PEGylation reactions of biomolecules.

The compositional heterogeneity associated with polymer conjugation reactions of biomolecules is analyzed for the particular case of nonspecific PEGylation reactions. It is shown that the distribution of the number of PEG moieties grafted to biomolecules such as proteins is a binomial-type function of two parameters-the reaction efficiency as well as the number of binding sites per biomolecule. The nature of this distribution implies that uniform compositions are favored for increasing number of coupling sites per biomolecule as well as for increasing efficiency of the modification process.

Understanding ligand distributions in modified particle and particlelike systems.

Chemical modification of nanoparticles or particlelike systems is ubiquitously being used to facilitate specific pharmaceutical functionalities or physicochemical attributes of nanocrystals, proteins, enzymes, or other particlelike systems. Often the modification process is incomplete and the functional activity of the product depends upon the distribution of functional ligands among the different particles in the system. Here, the distribution function describing the spread of ligands in particlelike systems undergoing partial modification reactions is derived and validated against a conjugated enzyme model system by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex.

Background: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem.

Isolation of mutations in the catalytic domain of the snf1 kinase that render its activity independent of the snf4 subunit.

Activation of the Snf1 kinase requires at least two events, phosphorylation of the activation loop on threonine 210 and an Snf4-dependent process that is not completely defined. Snf4 directly interacts with a region of the regulatory domain of Snf1 that may otherwise act as an autoinhibitory domain. In order to gain insight into the regulation of Snf1 kinase by Snf4, deletions in the regulatory domain of the catalytic subunit were engineered and tested for their effect on Snf1 function in the absence of Snf4.