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Biomedical subjects

R B Harris

Publications and source records attributed to R B Harris.

At least 127 records · Page 7Linked to original sources
PubMed1989

Growth hormone treatment reduces total body fat accumulation in Zucker obese rats.

Lean and obese Zucker rats were injected daily intraperitoneally with high doses (5-10 mg/kg) of human growth hormone (GH) for 3 weeks. In the obese rats after GH treatment, carcass lipid was decreased by 50 percent, and bone weight increased to levels of lean controls. During the last two weeks of GH treatment, food intake was increased in lean rats and not significantly affected in obese rats. Loss of body weight in obese animals was masked by water retention. Serum insulin concentrations were doubled in obese animals but unchanged in lean phenotypes after GH treatment. Hepatic fatty acid oxidation in obese animals was stimulated 5-fold by treatment, while hepatic lipid synthesis was stimulated 2-fold and adipose lipid synthesis was reduced 3-fold. These results suggest that growth hormone induces a partitioning of nutrients in obese rats which results in less lipid accumulation.

Adipose Tissue↗
PubMed1988

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor.

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.

Amino Acid Sequence↗
PubMed1988

The effects of adrenergic agonists and age on lipogenesis in avian hepatocytes.

1. Adrenergic inhibition of lipogenesis was examined in vitro using hepatocytes isolated from chickens 2-9 weeks old. 2. Lipogenesis was inhibited by beta 1, beta 2 and alpha 1 agonists. Greatest inhibition occurred when more than one type of receptor was stimulated. 3. Clonidine (alpha 2-agonist) may have stimulated lipogenesis. 4. Responsiveness to the agonists decreased as the chickens got older.

Aging↗
PubMed1988

Developmental changes in response to overfeeding: effect on composition of gain, liver metabolism and adipocyte cellularity in rats.

It has been proposed that voluntary food intake limits growth rate. The experiment described here investigated the effects of increased food intake on growth rate. Four age groups of female rats were given either 150 or 100 of ad libitum intake by oral gavage. Animals were tube-fed at these levels of intake for 1 wk. Overfeeding significantly increased carcass weight and fat for all ages examined. Carcass protein was significantly elevated in the overfed animals for the two youngest age groups. Differences between age groups in apparent energetic efficiency were small. Hepatic palmitate oxidation was depressed and esterification increased in all overfed rats. Fat cell size was increased by overfeeding in all age groups. In conclusion, the effects of overfeeding on the four age groups of rats studied were independent of age.

Adipose Tissue↗
PubMed1988

Voluntary food intake of lean and obese Zucker rats in relation to dietary energy and nitrogen content.

Genetically obese (fa/fa) Zucker rats maintain a normal lean body size but deposit excessive amounts of body fat. Preferential use of substrates for lipid synthesis results in inefficient use of dietary nitrogen for protein deposition. Three studies were carried out to determine whether an increased protein requirement caused hyperphagia and whether young Zucker rats preferentially regulated protein or energy intake. Rats were offered isoenergetic diets with nitrogen contents ranging from 4.5 to 53.2 mg/g, or isonitrogenous diets with energy contents of 2.2, 3.3 or 4.2 kcal/g. In both situations obese rats had significantly higher food intakes than lean rats. Within phenotype the rats maintained an almost constant energy intake so that nitrogen intake was proportional to dietary nitrogen concentration. In a third experiment lean and obese rats were given different proportions of their control protein intake by stomach tube. Energy intake was determined by voluntary consumption of a protein-free diet. Within phenotype energy intake was the same for all levels of protein intake. It appears that obese Zucker rats regulate energy intake at an elevated level and that protein intake is determined by dietary nitrogen content. Hyperphagia does not appear to result from a desire to obtain protein.

Animals↗
PubMed1988

Continuous fluorogenic substrates for atrial dipeptidyl carboxyhydrolase. Importance of Ser in the P1 position.

Several N-acyltetrapeptides of the general structure 2-aminobenzoyl-Gly-X-Phe(4-nitro)-Arg were synthesized and tested as substrates for atrial dipeptidyl carboxyhydrolase, an enzyme associated with atrial granules that converts one active atrial natriuretic peptide, atriopeptin II, to another, atriopeptin I. Hydrolysis of the X-Phe(4-nitro) bond generates the 2-aminobenzoyl fluorophore and the increasing fluorescence can be monitored in a continuous assay. Based on the ratio of Vmax/Km as an indication of substrate specificity, peptides containing X = Ser greater than Ala approximately equal to Lys- greater than Asn much greater than Thr approximately equal to Asp. With the exception of the Asn substrate, the Km determined for all the substrates was about the same. Thus, the effect of the P1 residue substitution shows up almost exclusively in Vmax.

Animals↗
PubMed1987

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme.

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Body composition of lean and obese Zucker rats in parabiosis.

Parabiosis is the surgical union of two animals to produce a chronic blood exchange. This model has previously been used to demonstrate the involvement of a blood-borne factor in the feedback control of food intake and regulation of energy balance. It has been hypothesized that obese rats produce a humoral agent that acts centrally to inhibit food intake and accumulation of fat. In this study 50-day-old male or female Zucker rats were joined in either lean-lean pairs or lean-obese pairs. They ate ad libitum until 152 days of age when body composition was determined. Parabiosis inhibited growth in all rats compared with single controls. Lean partners of obese rats had reduced carcass weights, the same percent body protein but less fat than members of lean-lean pairs. Female rats showed larger changes in body composition than did males. These results suggest that obese Zucker rats produce the hypothesized regulatory signal but do not respond to it.

Animals↗
PubMed1986

Isolation and sequencing of an active-site peptide from angiotensin I-converting enzyme.

A glutamic acid residue at the active-site of bovine lung angiotensin I-converting enzyme was esterified with p-[N,N-bis-(chloroethyl)amino]phenylbutyryl-L-[U-14]-Proline (chlorambucyl-L-[U-14C]-L-Proline), an affinity label for this enzyme. The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase HPLC contained the bound radiolabel. This active-site peptide (Mr approximately 16,000) was digested with trypsin, and the labeled peptide (T-2) was further degraded with thermolysin. The enzyme digest peptides were also resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained after thermolysin digestion (Th-1, Mr 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu. The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of PTH-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]-Proline which confirms our earlier findings. The sequence that we determined is homologous in five residues with the corresponding sequences of carboxypeptidase A and B, two other mammalian zinc-proteases. There is little sequence homology with thermolysin, a bacterial zinc-protease that also contains an essential active-site glutamic acid residue.

Amino Acid Sequence↗
PubMed1986

Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein.

A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.

Animals↗
PubMed1986

Influence of diet on the production of a "lipid-depleting" factor in obese parabiotic rats.

When one member of a parabiosed pair of rats is made obese its partner experiences a specific loss of body fat. In this study we determined whether production of the "lipid-depleting" factor in obese rats was diet specific. One member in each of 30 pairs was tube fed 200% normal intake of high carbohydrate (CHO), high fat corn oil or high fat medium-chain triglyceride diet. Their partners and both members of five control pairs ate CHO diet ad libitum. After 27 d of 200% feeding, in vitro hepatic, adipose and hypothalamic metabolism were measured. Composition of the diet used to induce obesity did not change the response of partners. All non-tube-fed partners had normal food intakes, body protein, hepatic fatty acid synthesis (FAS) and esterification (FAE), palmitate, glucose and beta-hydroxybutyrate oxidation. Adipose FAS and FAE were depressed, body fat was halved. Hypothalamic metabolism was not changed. A lipid-depleting agent originating in obese rats specifically inhibits adipose lipogenesis in their partners. Production of this factor does not appear to be influenced by dietary energy source.

Adipose Tissue↗
PubMed1986

Dynamics of recovery of body composition after overfeeding, food restriction or starvation of mature female rats.

In this study we examined the body composition of rats recovering from overfeeding, underfeeding or starvation. Female rats (220 g) were fed 160%, 100% or 40% of control intake, by stomach tube, until the 40% rats had lost 50 g. Other rats were starved to lose 50 g. Carcass composition was measured on one group from each treatment. The remaining rats returned to ad libitum feeding. The 160% rats were hypophagic and lost weight. Starved and 40% rats were hyperphagic and gained weight. Serum insulin increased with increased food intake. T4 was depressed by food restriction. T3 and T4 increased during weight loss in 160% rats. Carcass composition of rats from each treatment was determined at progressive stages of recovery. Overfed rats had gained 7 g of protein and 43 g of fat. Protein was soon lost but fat was still significantly increased after 44 d of recovery. Starved and restricted rats had lost 11 g of protein and 28 g of fat. Starved rats regained protein earlier than body fat. Restricted rats recovered body fat much earlier than body protein or weight. Body protein and fat may have individual regulatory mechanisms that work together to control body weight.

Animals↗
PubMed1986

Metabolic response to a specific lipid-depleting factor in parabiotic rats.

Parabiosis has been used as a technique for demonstrating the existence of a humoral factor in the control of body fat. The timing and metabolic basis for specific loss of fat from parabiotic partners of obese rats were examined. One member of a pair received 200% control intake, by stomach tube, for 8, 23, 39, or 57 days. Their partners ate 9.8 +/- 0.1 g/day. Members of ad libitum-fed pairs ate 9.6 +/- 0.1 g/day. All rats received the same diet. After 39 days, body fat in partners of obese rats was 6 +/- 1 g/rat compared with 17 +/- 1 g/rat in members of ad libitum pairs. Body protein was not different. In vitro hepatic fatty acid synthesis (FAS) and esterification (FAE) and inguinal FAS, FAE, and glycerol release suggested that fat loss was due to inhibition of adipose FAE. Partners of overfed rats and members of ad libitum pairs were then compared after 27 days of tube feeding when loss of fat was expected to be most rapid. Hepatic FAS, FAE, and fatty acid oxidation were the same for both groups. Inguinal FAS and FAE were decreased in partners of obese rats. An unidentified "lipid-depleting" agent, originating in obese rats, appears to inhibit adipose FAS and FAE in their partners independently of changes in feeding.

Adipose Tissue↗
PubMed1985

Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue.

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.

Amino Acid Sequence↗
PubMed1985

Comparison of hydrolysis of atriopeptin II stand-in substrate by atrial dipeptidyl carboxyhydrolase and angiotension I-converting enzyme.

We recently found and partially purified a new membrane-bound metallo dipeptidyl dipeptidase from bovine atrial tissue homogenates (Harris, R.B. & Wilson, I.B. (1984) Arch. Biochem. Biophys. 233, 667-675). We suggested that this enzyme was capable of cleaving the dipeptide, phenylalanyl-arginine from the C-terminus of atriopeptin II to give atriopeptin I. The atriopeptins are two atrial natriuretic peptides and the existence of the atrial peptide system has implicated the mammalian heart as an endocrine organ. The tetrapeptide benzoyl-glycyl-seryl-phenylalanyl-arginine was synthesized because it contains the C-terminal tripeptide sequence of atriopeptin II and should be useful to test the roles of the atrial enzyme and angiotensin I-converting enzyme in processing the atrial peptides. We found that for the atrial enzyme, Vmax was 13-fold higher and Km 7-fold-lower for this stand-in substrate than for benzoyl-glycyl-histidyl-leucine, a standard substrate used to measure converting enzyme activity. The ratio of Vmax/Km as a measure of substrate specificity indicates that the stand-in substrate is 86-fold better than benzoyl-glycyl-histidyl-leucine. In contrast, the stand-in substrate is a 20-fold poorer substrate for the converting enzyme than benzoyl-glycyl-histidyl-leucine. With the stand-in substrate, the converting enzyme showed pronounced substrate inhibition. An effective Vmax and Km were calculated using only concentrations of S below the optimum substrate concentration. These results confirm that the atrial enzyme is distinct from the converting enzyme. They also suggest that the conversion of atriopeptin II to atriopeptin I is a physiological process that is mediated by this enzyme.

Animals↗
PubMed1985

Level of satiety: fatty acid and glucose metabolism in three brain sites associated with feeding.

Two experiments were conducted to determine if the level of satiety alters fatty acid and glucose metabolism in selected brain areas. Rats received 150, 100, or 50% of normal intake by gastric intubation for 7 days. Thus the impetus for spontaneous feeding would be impaired in 150% fed rats (anoretic), potentiated in 50% fed rats (hungry), and maintained in 100% fed (control) rats. In vitro metabolism of glucose and palmitate was examined in liver, cortex, ventrolateral hypothalamus (VLH), ventromedial hypothalamus (VMH), and area postrema nucleus of the solitary tract. The VLH of hungry (50%) rats had a 40% increase in palmitate oxidation and a 9% decrease in glucose oxidation compared with controls (100%). The VLH of anoretic rats (150%) had a 36% decrease in palmitate oxidation and a 20% increase in glucose oxidation compared with controls. Hepatic metabolic differences were similar to those seen in VLH. Total fatty acid synthesis was 37% higher in anoretic and 29% lower in hungry compared with control rats. Recognition of excess, sufficient, or deficient peripheral energy status may be a process that monitors both VLH energy utilization and VMH energy storage in an attempt to depict metabolic adaptations of the periphery.

Animals↗