We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikrein. Four cloned overlapping double-stranded cDNAs gave a continuous mRNA sequence of 867 nucleotides beginning within the 5'-noncoding region and extending to the poly(A) tail. The mRNA sequence reveals that pancreatic kallikrein is synthesized as a prezymogen of 265 amino acids, including a proposed secretory prepeptide of 17 amino acids and a proposed activation peptide of 11 amino acids. The activation peptide, although similar in length, is distinct from those of the other classes of pancreatic serine proteases. The amino acid sequence of the predicted active form of the enzyme is closely related to the partial sequences obtained for other kallikrein-like serine proteases including rat submaxillary gland kallikrein, pig pancreatic and submaxillary gland kallikreins, the y subunit of mouse nerve growth factor, and rat tonin. Key amino acid residues thought to be involved in the substrate-cleavage specificity of kallikreins are retained. Hybridization analysis showed relatively high levels of kallikrein mRNA in the rat pancreas, submaxillary and parotid glands, spleen, and kidney, indicating the active synthesis of kallikrein in these tissues.Glandular kallikreins (EC 3.4.21.8) are members of a closely related subfamily of serine proteases that process polypeptide hormone precursors. Other members include the y subunit of nerve growth factor (1), the epidermal growth factor-binding protein (2), and tonin (3). Characteristically, each has a much more limited substrate-cleavage specificity than other serine proteases such as trypsin, chymotrypsin, and elastase. These kallikrein-like proteases cleave at one or a very few peptide bonds in their natural substrates with a general, but not absolute, preference for residues that have positively charged side chains and a strong bias for arginine over lysine (4,5
4).Kallikreins are found in many exocrine tissues, although their site(s) of synthesis remains unverified, and the nature and processing of presumed kallikrein precursors are unknown. Inactive precursor forms of kallikrein have been purified from rat (6) and porcine (7) pancreas. The precursors are acidic glycoproteins of apparent Mr 37,000. Slow activation occurs spontaneously and is accelerated by catalytic amounts oftrypsin. The activated enzymes are single polypeptides with slightly reduced molecular weight, suggesting the release of an activation peptide, which remains uncharacterized. Active kallikrein isolated from autolysed porcine pancreas consists of two polypeptide chains held together by disulfide bridges (8), indicative of further proteolysis.We report the identification and sequence analysis of the cloned mRNA sequence for rat pancreatic kallikrein and describe the nature of the preproenzyme and the presence of kallikrein mRNA in a number of rat tissues.
SynopsisOne hundred and fifteen patients from 5 general practices participated in a 12-week, double-blind study comparing L-tryptophan, amitriptyline, L-tryptophan–amitriptyline combination and placebo in the treatment of depression. Analysis of total score on the Hamilton Depression Scale and a global rating of depression showed that all 3 active treatments were more effective than placebo. Significantly more patients were withdrawn as treatment failures in the placebo group compared with the active treatment groups. Side-effects necessitated withdrawal of more patients from the amitriptyline group than from the other active tratment groups, but this difference was not significant. Plasma amitriptyline and nortriptyline levels were similar in the difference was not significant. Plasma amitriptyline and nortriptyline and biochemical profiles did not alter significantly in any group, but mean heart rate was significantly increased in patients receiving amitriptyline. There was no change in free or total plasma tryptophan concentration with treatment or on remission of symptoms.
It was shown previously that human placental trophoblastic cells use principally lipoprotein cholesterol for progesterone biosynthesis and that the rate of de novo synthesis of cholesterol is low. In addition, it was demonstrated that cholesterol derived from maternal plasma low density lipoprotein (LDL) rather than high density lipoprotein (HDL), is the principal source of placental cholesterol. In the present investigation, membrane fractions derived from human placenta were used to identify and characterize specific binding sites for both HDL and LDL. Pretreatment of membrane fractions with heparin resulted in an increase in the specific binding capacity for [125I]iodo-LDL 1.5 times that in membrane fractions not pretreated with heparin. Heparin pretreatment did not affect significantly the specific binding capacity of placental membranes for [125I]iodo-HDL. The specific binding capacity for [125I]iodo-LDL was 107 ng LDL protein mg-1 membrane protein, with an approximate Kd of 77 microgram LDL protein ml-1 in membranes pretreated with heparin. The specific binding capacity for [125I]iodo-HDL was much greater, equal to 323 ng HDL protein mg-1 membrane protein, with an approximate Kd of 152 microgram HDL protein ml-1. Each [125I]iodolipoprotein was specifically displaced by the corresponding respective nonradiolabeled lipoprotein. Preincubation of membranes with trypsin and pronase caused reductions in the specific binding capacity for [125I]iodo-LDL of 88% and 100%, respectively. Incubation of membranes with heparin caused displacement of [125I]iodo-LDL. However none of these treatments affected [125I]iodo-LDL. However none of these treatments affected [125I]iodo-HDL binding capacity. Similar binding sites for "125I]iodo-LDL and [125I]iodo-HDL were demonstrated in cells prepared from human placenta by trypsin digestion and maintained in monolayer culture.
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