Phosphorylated thiosugars: synthesis, properties, and reactivity in enzymatic reactions.

A number of phosphorylated thiosugars have been prepared and tested as substrates for metabolic reactions. 6-Thioglucose-6-P is readily synthesized by reaction of 6-tosylglucose with trisodium thiophosphate at pH 10 in aqueous solution; the product has only sulfur between carbon and phosphorus. When ethyl glycerate is tosylated and treated similarly with thiophosphate, a 5:1 mixture of 3-thioglycerate-3-P and the 2-isomer is formed. 6-Thioglucose-6-P is converted by glycolytic enzymes to triose phosphates, 3-thioglycerol-3-P and 3-thioglycerate-3-P, and is oxidized by enzymes of the hexose monophosphate shunt to 5-thioribulose-5-P, which can be converted via phosphoribulokinase and ribulose-bis-P carboxylase into 3-P-glycerate and 3-thioglycerate-3-P. For most of the non-phosphoryl-transferring enzymes there are only moderate effects on Vmax and Km. Phosphoglucoisomerase, however, is very sensitive to the sulfur for oxygen change, with Vmax decreasing 60-fold and Km increasing 15-fold. Surprisingly, phosphoribulokinase has a V/K value for 5-thioribulose-5-P that is over 3 orders of magnitude less than for ribulose-5-P. 6-Thio-glucose-6-P was found to be a substrate for several enzymes that transfer the phosphoryl group. It is as good a substrate for alkaline phosphatase as glucose-6-P, and with phosphoglucomutase it is converted to 6-thioglucose-1-P with a rate that is 11% of the rate of reaction of glucose-1-P, with a Keq value of 45.6. The free energy of hydrolysis of the phosphorylated thiol is thus -7.2 kcal/mol at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distortion of calculated whole-body hematocrit during lower-body immersion in water

We found a difference between the venous hematocrits of immersed and nonimmersed arms during immersion of the lower body in cold water but not during a comparable exposure to warm water. Fourteen healthy men were exposed to three different experimental conditions: arm immersion, body immersion, and control. The men always sat upright while both upper extremities hung vertically at their sides. During arm immersion, one forearm was completely immersed for 30 min in either cold water (28 degrees C, n = 7) or warm water (38 degrees C, n = 7). This cold-warm water protocol was repeated on separate days for exposure to the remaining conditions of body immersion (immersion of 1 forearm and all tissues below the xiphoid process) and control (no immersion). Blood samples were simultaneously drawn from cannulated veins in both antecubital fossae. Hematocrit difference (Hct diff) was measured by subtracting the nonimmersed forearm's hematocrit (Hct dry) from the immersed forearm's hematocrit (Hct wet). Hct diff was approximately zero when the men were exposed to the control condition and body immersion in warm water. In the remaining conditions, Hct wet dropped below Hct dry (P less than 0.01, 3-way analysis of variance). The decrements of Hct diff showed there were differences between venous hematocrits in immersed and nonimmersed regions of the body, indicating that changes of the whole-body hematocrit cannot be calculated from a large-vessel hematocrit soon after immersing the lower body in cold water.     

Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells.In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules.The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca²⁺ and MgATP. Although secretion requires Ca²⁺ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca²⁺ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides.These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca²⁺ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.We are grateful to Mr. John Gibbs for excellent technical assistance, and to the Medical Research Council (UK) for financial support.     

Antibiotic resistance indexing of Escherichia coli to identify sources of fecal contamination in water

A total of 202 Escherichia coli isolated from urban and rural water were tested with 11 antibiotics to assess the prevalence of antibiotic resistance from each source. Urban waters harbored higher percentages of resistant E. coli strains than rural waters. Antibiotic-resistant E. coli may offer an index of water quality related to source.     

Flexibility within the Heads of Muscle Myosin-2 Molecules

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN·nm/rad² is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor–lever junction.