Changes in the jugular haematocrit of sheep during feeding.

Intact and splenectomized sheep with and without a rumen fistula were used to investigate changes in the jugular blood haematocrit and plasma osmolality during hourly and once-daily feeding regimes. Osmolality was also estimated in the ruminal fluid of fistulated sheep with spleens. Haematocrit decreased in sheep with spleens before they were given a once-daily feed; it increased when these sheep started to feed, reaching a maximum increase of 13% after 30 min of feeding; it decreased during the remaining 45 min of feeding time and usually continued to decrease after feeding stopped. These changes were not due to diurnal influences. Splenectomized sheep fed once daily showed only small decreases in haematocrit before they were fed. Increases occurred with the onset of eating but they were smaller (7%) than in intact sheep and were of shorter duration. In hourly fed sheep with spleens, haematocrit decreased in the early stages of sampling in a manner similar to that for sheep fed once daily. The changes in haematocrit that did occur were not related in any obvious manner to the feeding regime. The haematocrit in splenectomized sheep fed hourly was stable throughout feeding. Variations in the haematocrit in splenectomized sheep, equivalent to a range of 13% in one of them, were observed in a series of blood samples obtained during a 5-h period remote from the feeding time. Large increases occurred in osmolality of ruminal fluid when sheep were fed daily and this was abolished by hourly feeding. Plasma osmolality in sheep fed once daily increased slowly. Maxima occurred after 100 min from the start of eating and were 7% greater than prefeeding values. Only minor changes were observed when these sheep were fed hourly.     

Dihydrofolate reductase from a methotrexate-resistant Escherichia coli: Proton magnetic resonance studies of complexes with folate and methotrexate

Proton magnetic resonance studies of 1:1 complexes of E. coli dihydrofolate reductase with folate and methotrexate were performed. A resonance at 1850 Hz in 1:1 enzyme-folate was assigned as the C-7 proton of bound folate by comparison with the spectra of enzyme complexed with folate specifically deuterated at C-7. The first order rate constant for folate dissociation was calculated to be less than 110 sec^?1. Four of the five histidine residues exhibited the same pK's and chemical shifts in the two complexes with pK values of 8.0, 7.3, 6.5 and ～5. However, one histidine increased its pK by 0.7 units (6.25→6.95) and its C-2 proton resonance shifted upfield 50 Hz when folate was substituted for methotrexate. Comparison of these results with those of chemical modification and ultraviolet difference spectroscopy experiments suggests that this histidine may be in the folate binding site — possibly near the pteridine portion of that site.     

Improved chemical synthesis and enzymatic assay of delta-1-pyrroline-5-carboxylic acid.

Δ¹-Pyrroline-5-car?ylic acid, an intermediate in both the biosynthesis and degradation ofl-proline, has been synthesized by the periodate oxidation of hydroxylysine and isolated as a pure compound, as indicated by enzymatic assay with pyrroline-5-car?ylate reductase fromEscherichia coli. Some features of the instability in solution ofΔ¹-pyrroline-5-car?ylic acid have been studied, leading to the conclusion that the rate of decomposition is sensitive to concentration of the compound. Colorimetric assay witho-aminobenzaldehyde was found to be an inadequate measure of the pyrroline compound in partially decomposed solutions.     

Hemoglobin-A2-Coburg or alpha2delta2116Arg leads to His (G18).

Hemoglobin-A2-Coburg or alpha2delta2-116 Arg leads to His (G18) has been found in members of a family of Sicilian origin. The propositus is heterozygous for hemoglobin-A2-Coburg as well as for beta-thalassemia, and family data indicate that the gene for the delta-Coburg chain is in trans of the beta-thalassemia determinant.     

Binding kinetics of mercury(II) TO POLYRIBONUCLEOTIDES.

Kinetic studies of the interaction of Hg(II) with polyribonucleotides have been used to investigate structural fluctuations of the bases in nucleic acids. The reaction of Hg(II) with poly(A)-poly(U) occurs in two phases which differ in time scale by a factor of about 100. The slow phase is first order and exhibits cooperativity or autocatalytic kinetics. The rate is found to increase as decreasing chain length of poly(U) is used to make the double helical complex. The reaction appears to initiate at the ends of poly(U) strands and may be associated with a molecular rearrangement which results in strand separation with Hg(II) being linked only to uridine. The fast reaction phase is second order ans shows little cooperative behavior. Protons are released at this stage indicating alteration of the double helix. The measured second-order rate constant is nearly three orders of magnitude smaller than that found for poly(U) alone. This rate difference suggests that the reactive sites are blocked by double helix formation, and become available for reaction with Hg(II) only through a structural fluctuation. The ratio of rate constants for the reaction of Hg(II) with poly(U) and poly(A)-poly(U) was used to place an upper limit on the equilibrium constant for the structural fluctuation of 2 times 10- minus 3 at 15 degrees and 0.5 M NaClO4. The heat of the "breathing" reaction can be estimated to be similar to 9 kcal/mol from comparison of the temperature coefficient of the reaction with poly(U) to that with poly(A)-poly(U).     

The two-step reversible denaturation of lactate dehydrogenase at low pH.

Upon exposure to conditions of low pH, beef B4 lactate dehydrogenase rapidly loses enzymatic activity, but this process can be completely reversed yielding 100% of the original activity if the enzyme is immediately returned to neutral conditions. As the time of exposure to low pH is increased, the fraction of activity recovered declines to a values of 50--60% and remains nearly constant over a period of many hours. Correlated with this behavior is a change in the kinetics of the recovery of activity. Recovery of activity has been shown to be a second-order process for enzyme exposed to low pH for brief periods of time (Anderson, S., and Weber, G. (1966), Arch. Biochem. Biophys. 116, 207). After several minutes at low pH recovery of activity is found to become first order and to occur at a considerably slower rate. Gel filtration chromatography at low pH separates the protein into two fractions. The lower molecular weight fraction is found to be primarily monomeric, as indicated by equilibrium ultracentrifugation, and is capable of recovering enzymatic activity. The higher molecular weight fraction is generated from the lower molecular weight fraction, and is incapable of recovering activity. These results are interpreted to indicate that the enzyme exists sequentially in three denatured forms at low pH, the first two capable of being restored to the native state, and the third irreversibly denatured.     

Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.