Chemical modification as an approach for the identification of UDPG-binding polypeptides of UDPG-glucose: (1,3)-Beta-glucan synthase

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5614146

Mason, T L

The lysine-reactive chemical modification reagents uridine diphosphate pyridoxal (UDP-pyridoxal) and formaldehyde (HCHO) were used to identify UDPG-binding polypeptides of UDP-glucose: (1,3)-{beta}-D-glucan synthase (GS) from red beet storage tissue. Complete enzyme inactivation occurred after exposure to micromolar levels of UDP-pyridoxal and millimolar levels of HCHO. Divalent cations (Mg{sup 2+} and Ca{sup 2+}, particularly Ca{sup 2+}) were required by both for inactivation. Substrate (UDPG) and chelators (EDTA and EGTA) protected plasma membrane GS (PMGS) against UDP-pyridoxal and HCHO inhibition. UDPG protected CHAPS solubilized GS (CSGS) against UDP-pyridoxal inactivation, but not against HCHO. It was concluded that beet GS contains a lysine residue at the UDPG-binding site. When PMGS was directly labeled with UDP({sup 3}H)-pyridoxal or ({sup 14}C)HCHO, random labeling occurred. Therefore, a multi-step labeling procedure was developed. Nonessential lysine residues were first blocked with HCHO while 5 mM UDPG protected the active site lysine. Background labeling was reduced 4-fold. Membranes were recovered by centrifugation and the active site lysine exposed to ({sup 14}C) HCHO. Major labeled polypeptides were at 200, 76, and 54 kD. Minor polypeptides were seen at 94, 82, 68, 60, and 20-25 kD. CSGS was labeled by a modified multi-step procedure. CSGS was blocked by reaction with UDP-pyridoxal in the presence of UDPG. CSGS was then recovered by product entrapment and labeled with ({sup 14}C)HCHO. Background labeling was reduced by 8-fold and potential UDPG-binding polypeptides narrowed to 68, 54, 25 and 22 kD.

• Research Organization: Rutgers--the State Univ., New Brunswick, NJ (USA)
• OSTI ID: 5614146
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALCOHOLS
ALDEHYDES
AMINO ACIDS
BEETS
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHELATING AGENTS
CHEMICAL COMPOSITION
EDTA
EGTA
ENZYME ACTIVITY
ENZYMES
FOOD
GLUCOSE
GLYCOLS
HEXOSES
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
INHIBITION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LABELLING
MAGNOLIOPHYTA
MAGNOLIOPSIDA
MEMBRANE PROTEINS
MEMBRANES
MONOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDES
PLANTS
POLYPEPTIDES
PROTEINS
REACTION KINETICS
REAGENTS
RECEPTORS
SACCHARIDES
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VEGETABLES