Effect of low ambient temperature on protein turnover and heat production in chicks

1. Effect of low ambient temperature on protein turnover in the liver and whole body was investigated in chicks together with the contribution of protein synthesis to the total heat production. 2. Both protein synthesis and degradation in the whole body were increased, the latter to a larger extent, at low ambient temperature (LT, 22 degrees C) compared with adequate temperature (AT, 30 degrees C). Liver protein synthesis was not significantly altered by the temperature treatment. 3. The total heat production of LT group was as high as 160% of the AT group. 4. The increased heat production due to enhanced whole-body protein synthesis accounted for only 1.4% of the heat increment in thermogenesis at low ambient temperature, suggesting that protein synthesis would contribute little, if any, to cold-induced thermogenesis in chicks.     

Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

Human skeletal muscle contains two major aminopeptidases: an anion-activated aminopeptidase B and an aminopeptidase M-like enzyme

Two major aminopeptidases, an aminopeptidase B and an aminopeptidase M-like enzyme, were purified from human skeletal muscle by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite column chromatographies. The purified aminopeptidase B exhibits a molecular weight of 76,000 under both native and denaturing conditions. The activity of the aminopeptidase B is regulated by C1 ions and other anions in vitro. On the other hand, the aminopeptidase M-like enzyme is a monomeric protein having a molecular weight of 96,000. It is capable of significantly cleaving Phe-, Leu-, Arg-, and Ala-aminoacyl bonds in the presence of 2-mercaptoethanol. The pH optima for both enzymes are around 7.0, and bestatin is an effective inhibitor of both enzymes.     

Three main components in plasma proteases and their relation to the renin-angiotensin system

The relation of plasma renin activity (PRA) and plasma levels of angiotensin I (AI) and II (AII) to those of various proteases, including eight endopeptidases and four aminopeptidases, was investigated in 51 normal control subjects. The multivariate study using factor analysis showed that the plasma proteases can be classified into three main components: the aminopeptidase, the plasmin, and the kinin-kallikrein. PRA and AI were related almost exclusively to the aminopeptidase component, while the AII level was related not only to the same component but also to the kallikrein-kinin component. This kind of multivariate study may help in the elucidation of the role of proteases and bioactive peptides, such as angiotensin derivatives, in essential hypertension through a comparison of multivariate relationships in controls and patients.     

Effects of two treatments on semen from different bulls on in vitro fertilization results of bovine oocytes

The semen of six different bulls was used to examine the effects of treatment with caffeine or caffeine plus Ca-ionophre on in vitro fertilization, cleavage and development into morulae of in vitro matured bovine oocytes. In vitro fertilization results (formation of both pronuclei, cleavage and development to >/= four-cell stage were significantly (P<0.01) higher using caffeine plus Ca-ionophre than those using only caffeine. The rates of fertilization and first cleavage were only slightly variable among the bulls. However, the present data showed significant variability in formation of both pronuclei (36 to 75%) of fertilized ova and development to the >/=4cell stage (39 to 71%) by different bulls. Development into morulae of ova recovered from the rabbit oviduct did not show any significant differences in relation to sperm treatments or individual bulls.     

Biochemical evidence for the local synthesis and retention of fibronectin in the tissue of human placenta

1. Human chorionic tissues were incubated with [14C]leucine and/or [3H]glucosamine, and fibronectin synthesis was examined. 2. Radio-labeled fibronectin was detected in the tissue fraction of the incubation mixture, but not in the medium fraction, indicating that fibronectin is synthesized and retained in the tissue. 3. The glycopeptides derived from 3H-labeled fibronectin showed the lectin-binding characteristics similar to those from unlabeled placenta fibronectin, but different from those of plasma fibronectin.     

Appearance and accumulation of c(4) carbon pathway enzymes in developing maize leaves and differentiating maize a188 callus

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C₄ photosynthesis, for cell morphology, and for ¹⁴C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later.

Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C₃ leaves and from etiolated C₄ leaf tissues, but differing from the corresponding enzymes in the mature leaf.

Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology.

Analysis of the labeled products of ¹⁴CO₂ fixation by nonchlorophyllous calli indicated β-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in β-carboxylation products was even greater. Analysis of enzyme levels in young leaf sections by protein blot and of ¹⁴C-labeling patterns in the present study are in general agreement with enzyme activity determinations of previous studies, providing additional information about PPDK levels, and supporting the model proposed for developing young leaves.

These results suggest that maize leaves begin to express C₄ enzymes during ontogeny through several stages from greening and cell differentiation as seen in the callus and then shoot formation, and finally acquire capacity for full C₄ photosynthesis during leaf development concomitant with the development of Kranz anatomy and accumulation of large amounts of enzymes involved in carbon metabolism.

     

Fucosylation of serum alpha-fetoprotein in patients with primary hepatocellular carcinoma

alpha-Fetoprotein specimens were prepared from the sera of four patients with hepatocellular carcinoma. The lentil lectin-reactive and lectin-nonreactive variants of this glycoprotein were also prepared from the serum of one of the four patients by affinity chromatography with immobilized lectin. The correlation between the carbohydrate structure of these compounds and their reactivity in crossed immuno-affinoelectrophoresis with lentil lectin was studied by chemical analysis and affinity chromatography of the glycopeptides with lectin columns. It was found that the lentil lectin-reactive variant contained a carbohydrate chain of the fucosylated biantennary complex type. These data together with previous findings indicate that most of the patients with hepatocellular carcinoma have an elevated serum concentration of fucosylated alpha-fetoprotein.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts

Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.

Location of PPDK in C₃ plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C₃ plant leaves as it is in the case of C₄ plants.