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Biomedical subjects

C Huang

Publications and source records attributed to C Huang.

At least 523 records · Page 29Linked to original sources
PubMed1980

Modification and identification of glutamate residues at the arginine-recognition site in the catalytic subunit of adenosine 3' :5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle.

It has been proposed that the active centre of cyclic AMP-dependent protein kinase contains an arginine-recognition site, which is considered to be essential for the function of the catalytic subunit of the kinase [Matsuo, Huang & Huang (1978) Biochem. J.173, 441-447]. The catalytic subunit can be inactivated by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide and glycine ethyl ester at pH6.5. The enzyme can be protected from inactivation by preincubation with histone, a protein substrate of the enzyme. On the other hand, ATP, which also serves as a protein kinase substrate, does not afford protection. Polyarginine, a competitive inhibitor of protein kinase, which is known from kinetic studies to interact specifically with the arginine-recognition site, partially protects the catalytic subunit from inactivation by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide. These results lead to the conclusion that the site of modification by carbodi-imide/glycine ethyl ester is most likely located at the arginine-recognition site of the active centre. A value of 1.7+/-0.2 (mean+/-s.d.) mol of carboxy groups per mol of catalytic subunit has been obtained for the number of essential carboxy groups for the function of protein kinase; a complete chemical modification of these essential carboxy groups results in total loss of catalytic activity. Finally, we have identified the essential carboxy group in the catalytic subunit of cyclic AMP-dependent protein kinase as being derived from glutamate residues. This is achieved by a three-step procedure involving an extensive proteolytic digestion of the [1-(14)C]glycine ethyl ester-modified enzyme and two successive high-voltage electrophoreses of the hydrolysate. It is concluded that 1.7mol of glutamyl carboxy groups per mol of catalytic subunit may be considered a component of the arginine-recognition site in the active centre of cyclic AMP-dependent protein kinase.

Amino Acids↗
PubMed1980

Kinetic studies on the dissociation of adenosine cyclic 3',5'-monophosphate from the regulatory subunit of protein kinase from rabbit skeletal muscle.

The exchange rate of unlabeled adenosine 3',5'-monophosphate (cAMP) with labeled [3H]cAMP in the dimeric regulatory subunit-cAMP complex of cAMP-dependent protein kinase, type I, purified from rabbit skeletal muscle is described by using the equilibrium isotope exchange technique. Results indicate that the rate of exchange carried out in the absence of the catalytic subunit (C) is rather slow with a half-life of approximately 870 s. This slow exchange rate is not affected by the presence of MgATP (50 microM). However, when both MgATP (50 microM) and C (1-13 NM) are present, the rate of isotope exchange is observed to increase markedly. Furthermore, less than stoichiometric amounts of C are required for the increase in the rate of cAMP exchange, indicating that the effect of C on the rate enhancement is a catalytic process. These results indicate that in the presence of MgATP, a ternary complex between C and regulatory subunit-cAMP complex must be formed, and a dynamic equilibrium between the eternary complex and its dissociable species must be reached within seconds. On the basis of our kinetic data, it is proposed that the formation of this ternary complex intermediate allows the rapid activation or the inactivation of cAMP-dependent protein kinase following changes in the cellular cAMP levels.

Animals↗
PubMed1980

Design of an artificial skin. II. Control of chemical composition.

Detailed methodology is described for the reproducible preparation of collagen--glycosaminoglycan (GAG) membranes with known chemical composition. These membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Such membranes have effectively protected these wounds from infection and fluid loss for over 25 days without rejection and without requiring change or other invasive manipulation. When appropriately designed for the purpose, the membranes have also strongly retarded wound contraction and have become replaced by newly synthesized, stable connective tissue. In our work, purified, fully native collagen from two mammalian sources is precipitated from acid dispersion by addition of chondroitin 6-sulfate. The relative amount of GAG in the coprecipitate varies with the amount of GAG added and with the pH. Since coprecipitated GAG is generally eluted from collagen fibers by physiological fluids, control of the chemical composition of membranes is arrived at by crosslinking the collagen--GAG ionic complex with glutaraldehyde, or, alternately, by use of high-temperature vacuum dehydration. Appropriate use of the crosslinking treatment allows separate study of changes in membrane composition due to elution of GAG by extracellular fluid in animal studies from changes in composition due to enzymatic degradation of the grafted or implanted membrane in these studies. Exhaustive in vitro elution studies extending up to 20 days showed that these crosslinking treatments insolubilize in an apparently permanent manner a fraction of the ionically complexed GAG, although it could not be directly confirmed that glutaraldehyde treatment covalently crosslinks GAG to collagen. By contrast, the available evidence suggests strongly that high-temperature vacuum dehydration leads to formation of chemical bonds between collagen and GAG. Procedures are described for control of insolubilized and "free" GAG in these membranes as well as for control of the molecular weight between crosslinks (Mc). The insolubilized GAG can be controlled in the range 0.5--10 wt. % while "free" GAG can be independently controlled up to at least 25 wt. %; Mc can be controlled in the range 2500--40,000. Studies by infrared spectroscopy have shown that treatment of collagen--GAG membranes by glutaraldehyde or under high-temperature vacuum does not alter the configuration of the collagen triple helix in the membranes. Neither do these treatments modify the native banding pattern of collagen as viewed by electron microscopy. Collagen--GAG membranes appear to be useful as chemically well-characterized, solid macromolecular probes of biomaterial--tissue interactions.

Amino Acids↗
PubMed1979

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.

Animals↗
PubMed1978

Geometric packing constraints in egg phosphatidylcholine vesicles.

This investigation presents a quantitative analysis of the asymmetric transmembrane lipid packing geometry in egg phosphatidylcholine vesicles. The analysis uses hydrodynamic and nuclear magnetic resonance data obtained for homogeneous egg phosphatidylcholine vesicles and demonstrates that the average area per lipid head group and effective length of the lipid are greater on the outer monolayer than on the inner monolayer of the egg phosphatidylcholine vesicle. Results also indicate that the bilayer of the egg phosphatidylcholine vesicles has an asymmetrical interface through its center.

Animals↗
PubMed1977

Phospholipid head-group conformations; intermolecular interactions and cholesterol effects.

The predominant orientation of the phosphorylcholine polar head group in phosphatidylcholine and sphingomyelin bilayers and cholesterol perturbations of that orientation have been identified by exploiting the 31P (1H) nuclear Overhauser effect (NOE) in the 31P NMR spectra of phospholipid bilayers. In pure egg phosphatidylcholine bilayers, a NOE of 40% is observed. The magnitude of the NOE has been measured as a function of continuous-wave proton-decoupler frequency in order to identify the proton source of the NOE. In pure egg phosphatidylcholine bilayers, the maximum NOE occurs at the N-methyl proton resonance position of the choline moiety. In a modified phosphatidylcholine in which all the N-methyl protons were replaced by deuterium, the NOE arose from methylene protons next to the phosphate. In mixed systems of phosphatidylcholine and phosphatidylethanolamine, and phosphatidylcholine and diphosphatidylglycerol, both phospholipid resonances attained maximum NOE at the position of the N-methyl proton resonance of phosphatidylcholine. An analogous result was obtained with pure sphingomyelin. These results are explained by orienting the phosphorylcholine portion of the molecule parallel to the surface of the bilayer so that the positively charged N-methyl moiety is located close to the negatively charged phosphate on a neighboring phospholipid in an intermolecular interaction. Addition of cholesterol is shown to disrupt the intermolecular interaction in phosphatidylcholine bilayers.

Chemical Phenomena↗
PubMed1977

Mechanochemical studies of enzymatic degradation of insoluble collagen fibers.

A mechanochemical method was developed for studying the enzymatic degradation of insoluble collagen fibers. The method involves stretching the collagen fiber to a fixed extension in the presence of a solution of collagenase and measuring the rate of relaxation of the force induced in the fiber. In this work, bacterial collagenase was used for reasons of availability. We observed invariably an exponential decrease in force with respect to ttime. The slope of the linear plot of logarithm of the force versus time was taken as a measure of the rate of enzymatic degradation. This rate was found a) to vary linearly with collagenase concentration; b) to be maximal at pH 7-8; c) to vary with temperature according to the Arrhenius relationship in the range 10-56 degrees C; d) to be reduced to varying extent by addition of EDTA omicron-phenanthroline, 2,3-dimercaptopropanolol, and D,L-cysteine; e) to be minimal when the strain on the fiber was ca. 4%; f) to be increased dramatically by denaturation of the collagen fiber; and g) to be reduced by an increase in the crosslink density of the collagen fiber. Except for the effect of strain, which can not be conveniently studied by existing methods these results are consistent with those observed by other methods for the study of the enzymatic degradation of collagen. The mechanochemical method is, however, uniquely suited to monitor continuously the enzymatically induced decay in the stress-bearing ability of collagen fibers. It has also been found useful in the design of collagenous implants with specified resistance to enzymatic degradation in vivo.

Collagen↗
PubMed1977

Configurations of fatty acyl chains in egg phosphatidylcholine-cholesterol mixed bilayers.

Based on the structural properties of cholesterol and egg phosphatidylcholine, and on the assumption that the van der Waals' type attactive interaction between the steroid nucleus and the fatty acyl chains provides a stabilizing force for the cholesterol-egg phosphatidylcholine complex, some specific orientation and configurations of the fatty acyl chains around the steroid nucleus in the interacting system are proposed in terms of an optimal packing. The proposed model suggests thathe saturated chains are largely facing the flattened (alpha) surface of the steroid nucleus of cholesterol, while the unsaturated chains can interact with both the alpha and beta surfaces of the steroid nucleus. It is also suggested that the angular methyl groups on the beta surface of the steroid nucleus lock the unsaturated fatty acyl chain in a relatively immobile configuration. Experimental evidence which provides support for the proposed stereochemical model is presented.

Chemical Phenomena↗
PubMed1977

Vitamin E status of Chinese population in Taiwan.

Vitamin E status of Chinese population with different age groups was studied by determining plasma vitamin E levels (PE) and hydrogen peroxide-induced erythrocyte hemolysis (HPEH). The mean PE of 99 adults, ages 23 to 78, was 1.05 +/- 0.47 mg/100 ml. There was no significant difference between sexes. PE was negatively correlated with HPEH (P less than 0.01). There was a significant positive correlation (P less than 0.01) between PE and plasma cholesterol level. The mean PE of four young men of Yami tribesmen, ages 16 to 19, was 0.60 +/- 0.10 mg/100 ml. HPEH was low. After daily supplementation of 200 mg dl-alpha-tocopheryl acetate for 16 days, PE increased 54%, but the plasma cholesterol was not affected. The mean PE of 39 elementary school children ages 12 to 13, was 0.70 +/- 0.31 mg/100 ml. The mean PE of 20 newborn infants was 0.23 +/- 0.08 mg/100 ml. HPEH was as high as 56.5 +/- 31.9% indicating a state of vitamin E deficiency. PE of total population was positively correlated with age (P less than 0.01). It was concluded that the vitamin E status of Chinese population in Taiwan was comparable to that of population groups in many other parts of the world.

Adolescent↗