Collagen fibrillogenesis in vitro: an investigation of the thermal memory effect and of the early events occurring during fibril assembly using dynamic light scattering

Dynamic light scattering has been used to characterize a variety of lathyritic rat skin collagen solutions. The technique was used to monitor the onset of fibril assembly in vitro and to investigate the thermal memory effect. Although the incorporation of thermal memory was demonstrated by reheating the sample and subsequently observing a shortened turbidimetric lag phase, no significant differences between naive solutions and ones exhibiting thermal memory could be detected using photon correlation spectroscopy. This suggests that subtle changes in the state of the collagen molecules rather than extensive changes in the degree of aggregation are responsible for the thermal memory effect. During fibrillogenesis, no large-scale changes in the distribution of monomers or aggregates occur until near the end of the lag phase.     

Genetic analysis of cystic fibrosis: linkage of DNA and classical markers in multiplex families

Linkage of cystic fibrosis (CF) to DNA and classical markers was studied in 36 families of two or three generations with at least two living affected children. Among the 79 affected children, no recombinants were detected between the disease and the markers MET and pJ3.11, previously shown to be linked to CF. No linkage between the human trypsin gene family (which appears to include at least 10 members) and CF was found, although not all genes of the trypsin family have been screened yet. In one of the CF families, recombination between MET and pJ3.11 was detected in an unaffected sib. Data from our families suggest that the gene order of markers among chromosome 7q is: (7cen;p8.33)collagen(COL1A2);DOCR1-917;paraoxonase+ ++(PON);(MET-cf-J3.11);T-cell receptor beta chain (TCRB);qter. There was no evidence for (or against) either postzygotic selection or meiotic drive to explain the high frequency of CF in Caucasian populations.     

Polymorphisms of mitochondrially encoded proteins.

Polymorphisms of mitochondrially encoded proteins can be detected in human lymphocytes by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Using an SDS-polyacrylamide 8 M urea system, 17 mitochondrially encoded proteins are distinguishable. Three of these (ME-6, ME-8, and ME-17) were polymorphic among 92 individuals screened, and these polymorphisms are reported here for the first time. With SDS-polyacrylamide electrophoresis without urea, 18 mitochondrial proteins are detectable. One of these (MV-1) varied in two of 31 individuals tested. This polymorphism has been identified previously in HeLa cells. Maternal inheritance of the ME-8 polymorphism was demonstrated by three informative families.     

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.     

Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347

T7 RNA polymerase has been purified to homogeneity from an overproducing clone of Escherichia coli containing pAR1219. Preparations have a zinc content as low as 0.01 mol/mol of enzyme and a high specific activity, 300 000-500 000 units/mg. There are no intrinsic zinc sites. Furthermore, extrinsic Zn2+ does not function as an activator. Supplementation of the assay mix with up to 5 mM ethylenediaminetetraacetic acid has little effect on activity while added Zn2+ is strongly inhibitory at concentrations above 10 microM. This monomeric RNA polymerase is not a zinc metalloenzyme, unlike its multimeric bacterial counterparts. Titration of the urea-denatured protein with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that all 12 Cys residues are present in the free sulfhydryl form, 5 of which are readily accessible to reagent in the native enzyme. More preferential labeling of the sulfhydryls can be achieved with low concentrations of [14C]iodoacetamide, where inactivation of the enzyme proceeds with incorporation of approximately 1.2 mol of [14C]iodoacetamide/mol of polymerase. Amidomethylation primarily occurs at Cys-347, with lesser reaction at Cys-723 and Cys-839. Cys-347 and Cys-723 are in segments of the primary sequence containing numerous basic residues. These same segments have previously been implicated in promoter binding, suggesting that both residues are located within or near the active site region.     

Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes

Removal of lipid from detergent-solubilized succinate cytochrome c reductase by a mild method leads to a series of changes in the optical and EPR spectra of the b cytochromes. This culminates in a state that resembles purified b cytochromes from the same source and bisimidazole ferriheme model complexes. Reconstitution of the lipid-depleted complex with phospholipid restores the native spectra in a significant fraction of the complexes in the early stages of lipid depletion. Once the final state has been reached, however, reconstitution has so far been incapable of restoring described in this communication can be related to a model for integral membrane cytochromes.     

Adrenergic and local control of O2 uptake during and after severe hypoxia

The distribution of whole-body O2 supply during severe hypoxia and recovery and its relation to the regional distribution of O2 deficit and repayment was studied. Mongrel dogs were anesthetized, paralyzed, and ventilated to maintain an end-tidal PCO2 between 35 and 40 Torr. In one group, the alpha- and beta-adrenergic receptors were blocked to eliminate neural and humoral adrenergic influences. In a second group, alpha-adrenergic receptors were stimulated to decrease O2 delivery by excessive vasoconstriction. In a third group, beta-adrenergic receptors were stimulated to increase O2 delivery. Whole-body and hindlimb muscle O2 uptake and vascular responses were measured during normoxic control, 15 or 30 min of severe hypoxia (9% O2 in N2), and 20 or 30 min of normoxic recovery, respectively. The whole-body O2 deficit and excess O2 uptake in recovery were partitioned into muscle and nonmuscle areas. The data showed that neural or humoral influences had little effect on the regional distribution of the total O2 deficit and O2 excess in recovery. The O2 deficit could be decreased somewhat by increasing delivery, but the amount of excess O2 used in recovery was unaffected. This suggested that the excess O2 use in recovery was more a function of an energy deficit during hypoxia and not an O2 deficit.